Polyunsaturated fatty acid derivatives and their use

ABSTRACT

Novel amides are disclosed, in particular amides of the all-trans-retinoic acid or 13-cis-retinoic acid and arachidonic acid and docosahexaenoic acid and eicosapentaenoic acid or linoleic acid with 2-aminoethanol, alpha-L-serine, alpha-L-threonine, alpha-L-tyrosine containing phosphate groups. Further, the present invention discloses the synthesis and use of these compounds, in particular their pharmaceutical application.

This is a Divisional application of U.S. patent application Ser. No.09/471,105 filed Dec. 21, 1999 now U.S. Pat. No. 6,274,747.

FIELD OF THE INVENTION

This invention relates to novel polyunsaturated fatty acid derivativesand their use as therapeutic agents.

BACKGROUND OF THE INVENTION

Amides of fatty acids (saturated and unsaturated) having the followingformula:

R—CO—NH₂

wherein the R is alkyl residue of fatty acid, represent a new group oflipid bioregulators originated from long-chain fatty acids via amidationby corresponding amines. Erucamide (13-docosenamide) was found to be themajor bovine mesentery angiogenic lipid. The mechanism of angiogenicactivity is unknown and this lipid does not promote proliferation ofendothelial cells or induce inflammatory effects (Wakamatsu K. et al.,Biochem. Biophys. Res. Commun., V.168. p. 423-429, 1990). A moleculeisolated from the cerebrospinal fluid of sleep-deprived cats has beenchemically characterized and identified as cis-9,10-octadecenoamide.Other fatty acid primary amides in addition to cis-9,10-octadecenoamidewere identified as natural constituents of the cerebrospinal fluid ofcat, rat and human, indicating that these compounds compose a distinctfamily of brain lipids. Synthetic cis-9,10-octadecenoamide inducedphysiological sleep when injected into rats. Together, these resultssuggest that fatty acid primary amides may represent a previouslyunrecognised class of biological signal molecules (Cravatt B. F. et al.,Science., V. 268., P.1506-1509, 1995).

A very thoroughly investigated group of compounds are ethanol amides offatty acids, having the following formula:

R—CO—NH—CH₂CH₂—OH

where R is an alkyl residue of a fatty acid.

Ethanol amides of fatty acids bound with cannabinoid receptors in thecentral nervous system or in peripheral tissues are considered asendogenous ligands for these receptors. They exhibit pharmacologicalpatterns like cannabimimetics (Bezuglov V. V. et al. Biochemistry(Moscow). V.63, N 1. P. 27-37,1998).

Amides of retinoic acid (cis-trans isomers) with some amino acids havingthe following formula:

R—CO—NH—Y—COOH

wherein R is an alkyl residue of retinoic acid and Y is a residue ofamino acid have been synthesised via all-trans-retinoyl chloride and anester of the amino acid (Shealy Y. F. et al., J.Med.Chem.V.31.,P.190-196, 1988). The retinoyl derivatives of leucine,phenylalanine, alanine, tyrosine and glutamic acid were prepared. The13-cis-retinoyl derivatives of leucine, phenylalanine, alanine, andglycine were prepared similarly from 13-cis-retinoic acid. In assays ofthe retynoylamino acids for reversal of squamous metaplasia in hamstertrachea organ cultures, these compounds were less active than retinoicacid, but the leucine, alanine and phenylalanine derivatives weresimilar in activity to several retinamides that suppress bladdercarcinogenesis in vivo. Two of the retinoylamino acids, as well as twosimple retinamides were shown to be moderately cytotoxic to murineleukemia and human epidermoid carcinoma cells in culture.

One problem addressed by the present invention is that of makingavailable novel compounds for use in the manufacture of pharmaceuticals,e.g. for the treatment of cancer and immune deficiencies, said compoundsexhibiting i.a. higher activity than presently known compounds.

SUMMARY OF THE INVENTION

The present invention makes available novel amides according to theattached claims, in particular amides of the all-trans-retinoic acid or13-cis-retinoic acid and arachidonic acid and docosahexaenoic acid andeicosapentaenoic acid or linoleic acid with 2-aminoethanol,alpha-L-serine, alpha-L-threonine, alpha-L-tyrosine containing phosphategroups. Further, the present invention discloses the use of thesecompounds, in particular their pharmaceutical application as specifiedin the attached claims.

The structure of these compounds is covered by the following generalformula:

 R—CONH—X—OPO(OH)₂

wherein

R is

or CH₃(CH₂)₄(CH═CH—CH₂)₄CH₂CH₂—

or CH₃(CH₂CH═CH)₆CH₂CH₂—

or CH₃(CH₂CH₂CH═CH)₅(CH₂)₃—

or CH₃(CH₂CH═CH)₃(CH₂)₇—

and X is —CH₂—CH₂—

or —CH(CO₂H)—CH₂—

or —CH(CO₂H)—CH(CH₃)—

or —CH(CO₂H)—CH₂—C₆H₅—

Thus the amino group of 2-aminoethanol or the alpha-amino group of aminoacid forms an amide bond with the carboxylic group of arachidonic acid,docosahexaenoic acid, eicosapentaenoic acid or linoleic acid andall-trans-retinoic acid or 13-cis retinoic acid. At the same time thehydroxyl group of 2-aminoethanol and the amino acid is modified by aphosphate residue. The compounds can be applied as immunostimulatingtherapeutical agents for the treatment of immune-deficiencies.

DESCRIPTION OF THE INVENTION

The novel compounds according to the present invention are amides ofall-trans-retinoic acid or 13-cis-retinoic acid and arachidonic acid anddocosahexaenoic acid and eicosapentaenoic acid or linoleic acid with2-aminoetanol, alpha-L-serine, alpha-L-threonine, alpha-L-tyrosine. Atthe same time hydroxyl groups of amino acids and 2-aminoethanol aremodified by phosphate residues. The all-trans-retinoic acid or 13-cisretinoic acid and arachidonic acid and docosahexaenoic acid andeicosapentaenoic acid or linoleic acid have been derived by variousprocedures from naturally-occurring products. It is however possible,within the scope of the present invention, to produce these compoundsynthetically.

The main characteristic among the novel synthesised compounds is thephosphorylation of the hydroxyl groups of N-acyl derivatives of aminoacids and 2-aminoethanol.

Retinoic acid derivatives according to the present invention include thefollowing compounds:

1. N-(all-trans-retinoyl)-o-phospho-2-aminoethanol

1a. N-(13-cis-retinoyl)-o-phospho-2-aminoethanol

2. N-(all-trans-retinoyl)-o-phospho-L-serine

2a. N-(13-cis-retinoyl)-o-phospho-L-serine

3. N-(all-trans-retinoyl)-o-phospho-L-threonine

3a. N-(13-cis-retinoyl)-o-phospho-L-threonine

4. N-(all-trans-retinoyl)-o-phospho-L-tyrosine

4a. N-(13-cis-retinoyl)-o-phospho-L-tyrosine

Arachidonic acid derivatives according to the present invention includethe following compounds:

5. N-arachidonoyl-o-phospho-2-aminoethanol

6. N-arachidonoyl-o-phospho-L-serine

7. N-arachidonoyl-o-phospho-L-threonine

8. N-arachidonoyl-o-phospho-L-tyrosine

Docosahexaenoic acid derivatives according to the present inventioninclude the following compounds:

9. N-docosahexaenoyl-o-phospho-2-aminoethanol

10. N-docosahexaenoyl-o-phospho-L-serine

11. N-docosahexaenoyl-o-phospho-L-threonine

12. N-docosahexaenoyl-o-phospho-L-tyrosine

Eicosapentaenoic acid derivatives according to the present inventioninclude the following compounds:

13. N-eicosapentaenoyl-o-phospho-2-aminoethanol

14. N-eicosapentaenoyl-o-phospho-L-serine

15. N-eicosapentaenoyl-o-phospho-L-threonine

16. N-eicosapentaenoyl-o-phospho-L-tyrosine

Linoleic acid derivatives according to the present invention include thefollowing compounds:

17. N-linolenoyl-o-phospho-2-aminoethanol

18. N-linolenoyl-o-phospho-L-serine

19. N-linolenoyl-o-phospho-L-threonine

20. N-linolenoyl-o-phospho-L-tyrosine

The structural formulas of these compounds are presented below:

In developing the scheme of synthesis the present inventor took intoaccount the following properties of the title compounds:

i) a high degree of unsaturation

ii) presence of an asymmetric centre

iii) presence of sufficiently labile groups e.g. amide and phosphate.

Many of the well-known methods of acylation and phosphorylation couldtherefore not be used for the synthesis of such compounds. Thepreparation of the mixed carbonic carboxylic anhydrides followed byacylation of amino moiety of the methyl esters of hydroxyamino acids andethanolamine was carried out at low temperature. The method of synthesisdeveloped by the present inventor enables the N-acyl derivatives to beobtained with nearly quantitative yields. It is demonstrated thatbeta-cyanoethyl phosphate is a universal phosphorylation agent forhydroxyamino acids containing primary, secondary or aromatic OH-groups.Beta-cyanoethyl and ester protective groups were removed simultaneouslyby mild alkaline hydrolysis (0° C., 1.5 N NaOH). It is worth emphasisingthat all compounds prepared by this method are formed in good orreasonable yields. In all experiments, the inventor has used synthesisedtitle compounds in the form of ammonia salts.

The molecules of the title compounds have a polar and non-polar part(hydrophilic and hydrophobic in water systems) simultaneously. Theselong chain molecules are present in water solutions in the form ofmicelles, if their concentration is equal to the critical micelleconcentration or higher. The structural organisation of the titlecompounds in micelles could be considered as a new organisation level ofbiological active molecules with specific physiological activity. Itshall be noted that the process of micelle formation is reversible.

The influence of the title compounds 1 through 4, 1a through 4a, and 5through 20 on humoral immune response was estimated by counting thequantity of antibody-forming cells (AFC) in the mice spleen. It has beenexperimentally proved, that the immunogenic activity of all compounds isdose-dependent. In particular, compounds 8, and 10 through 14, 17, 19and 20, in doses of 45.5 and 91 μg/mouse exhibit no immunogenicactivity, but at the same time in the dose of 136.5 μg/mouse thesecompounds displayed an immunostimulating action. It was found that therelative amount of AFC on the 5^(th) day after injection of compound 10increased by 138% and the total AFC amount increased by 159%. Forcompound 11—an increase of 67% and 80%, respectively, was recorded. Forcompound 12—an increase of 134% and 99% respectively, was recorded. Forcompound 8—an increase of 31% and 91% respectively, was recorded. Forcompound 13—an increase of 41% and 49% respectively, was recorded, andfor compound 14—an increase of 52% and 51% respectively. For compound17, the increase was 31% and 30%; for compound 19, 40% and 40%; forcompound 20, 49% and 51%, respectively. The increase was calculated ascompared with the amount of cells in the animals immunised only withsheep erythrocytes.

Compounds 3, 4, 1a, and 2a, in the doses of 45.5 μg/mouse, exhibit noimmunogenic activity. It was found that the relative amount of AFC onthe 5^(th) day after injection to the animals, for the compound 3, in adose of 91 and 136.5 μg/mouse, increased by 103% and 136%; and inrespect of the total AFC amount an increase of 49%; and 164%,respectively, was recorded. For compound 4 in a dose of 91 and 136.5μg/mouse, the relative amount of AFC increased by 70% and 233% and thetotal AFC amount increased by 45% and 130%, respectively. For compound1a in a dose of 91 and 136.5 μg/mouse, the relative amount of AFCincreased by 29% and 52%, and the total AFC amount increased by 27% and66%, respectively. For compound 2a in a dose of 91 and 136.5 μg/mouse,the relative amount of AFC increased by 93% and 111%, and the total AFCamount increased by 48% and 71%, respectively. The increase iscalculated as a comparison with the amount of cells in animals immunisedonly with sheep erythrocytes.

The critical micelle concentration (CMC) values for compounds 1 through4, 1a through 4a were determined as described by Griess W. (Fette SeifenAnstrichmi., 1955. Bd.57, s. 24-32) and for compounds 5 through 20 theCMC values were determined as described by A. Chattopadhyay and E.London (Anal. Biochem., 1984, 139, P.408-412). The CMC-values forammonia salts of compounds 1 through 4 and 1a through 4a are in theinterval of 1·10⁻⁴ M-4·10⁻⁴ M and for ammonia salts of compounds 5through 20, in the interval of 1·10⁻³ M-2·10⁻³ M. That explains why, inthe present experiments, compounds 1a, 2a, 3, and 4 displayimmunostimulating effect in vivo in micelle form at 91 μg/mouse andcompounds 8, and 10 through 14, 17, 19 and 20—at 136.5 μg/mouse.

According to the obtained results, the title compounds displayimmunostimulating effects in vivo resulting in the enhancement of theamount of antibody-forming cells (AFC) in the spleen and in theenhancement of the antibody titres. Some of these compounds induce theconversion of B-lymphocytes to AFC and enhance the production ofIgM-antibodies, ensuring a 2 to 3-fold increase of the immune responsein C57B1/6 mice which are low-reactive to sheep red blood cells (SRBC).

It is known that one of the factors of genetically determined lowreactivity of some lines of mice is insufficient extent of the processesof T- and B-lymphocyte migration and co-operation in immunisation withthe particular antigen. Thus, using the studied compounds made itpossible to convert genetically low-reacting animals to high-reactingones. Lymphokines and monokines such as interleukin-1 and -2, thymichormones, hematopoetin, factor of tumour necrosis, interferons, themolecular weight of which is less then that of albumin, are capturedactively by the liver, filtrated by the kidneys, which results in theirquick disappearance from the blood stream. The interest attracted bythese immunomodulators is not too great due to their low effectivenessand high toxicity. The studied compounds can be promising as potentmeans for correction of immunity disorders localised at the level ofimmunocompetent cells, including the possibility of phenotypiccorrection of the immune response.

Reversible protein phosphorylation is one of the most importantmechanisms of regulating intracellular processes such as cell cycle,growth and differentiation (Edelman A. M. et al., A. Rev. Biochem. V.56., P. 567-613, 1987; Han K.-K., Martinage A., Int. J. Biochem. V. 24.N1.,P. 19-28, 1992). The most common phosphorylated amino acids areserine, threonine and tyrosine (on the hydroxyl group). Calcium and cAMPexert many of their cellular effects by controlling the activity of aprotein kinases. Protein kinases catalyse the transfer of phosphategroups from a molecule of adenosine triphosphate (ATP) to otherproteins. The addition of a phosphate group alters protein function;indeed, widespread protein phosphorylation is thought to underlie thechanges in cell behaviour induced by some extracellular signals. It iscontemplated by the present inventor that the title compounds could besuch signal molecules.

Alpha-fetoprotein (AFP) forms a reversible equilibrium complex withN-arachidonoyl aminoethylphosphate (N-AAP). The protein isreversiblylinked with to a micelle containing up to 300 molecules ofN-AAP (See International Application No PCT/EP99/04201 by the sameinventor).

The inventor has now shown that AFP forms reversible equilibriumcomplexes with all the title compounds of the present invention. Theseinventive complexes may contain their components in highly varying molarratios, such as from an equimolar ratio to a significant overabundanceof compounds 1 through 4, 1a through 4a, and 5 through 20 in relation toAFP. For compounds 1 through 4, 1a through 4a, and 5 through 20, theinhibition equilibrium association constants (K_(i)) of arachidonic acidwith AFP were determined to be in the interval—0.9·10⁶ M⁻¹-4·10⁶ M⁻¹.

The inventive complexes may be obtained by adding water solution of anytitled compound to a water solution of AFP followed by ultrafiltration,said filtration resulting in concentrating the solution and removing anycompound (e.g. any one of compounds 1 through 4, 1a through 4a, 5through 20) that remained unbound to AFP. The AFP concentration insolution varies from 0, 1 to 2 mg/ml and that of the compound from 0,005up to 30 mg/ml. The changes of molecular weight of the AFP as judged bygel-filtration is an evidence of the existence of AFP complexes withmicelles of compounds 1 through 4, 1a through 4a, and 5 through 20.

In one embodiment the molecular weight of AFP incorporated in thecomplex with the title compound increases by approximately 2 times.Micelles contained about 100-200 lipid molecules. In another embodimentthe molecular weight of AFP incorporated in the complex with any one ofthe compounds 1 through 4, 1a through 4a, and 5 through 20 increases byapproximately 2-3 times. The micelles contained 200-300 lipid molecules.

The influence of complexes of AFP with one of the title compounds 1-4,1a-4a, 5-20 as well as their basic components on humoral immune responsewas estimated by counting the quantity of antibody-forming cells (AFC)in the mice spleen. In particularly it has been experimentally provedthat a compounds 1, 2a, 3 in the dose of 45.5 μg/mouse and a compounds7, 10, 14, 15 in the dose 91 μg/mouse and human or rat AFP in the dose 9μg/mouse do not inhibit immunogenic activity.

Administration of a complex of AFP with compound 1 in the dose of 45.5μg/mouse (complex 1:100) resulted in that the relative amount of AFCincreased by 56% and the total AFC amount increased by 43%; for complexAFP with compound 2a in the dose of 45.5 μg/mouse (complex 1:100) thecorresponding figures increased by 77% and 43% accordingly; for acomplex APP with compound 3 in the dose of 45.5 μg/mouse (complex 1:100)the corresponding figures increased by 76% and 48% accordingly incomparison with the amount of cells in animals immunized only with sheeperythrocytes.

Administration of a complex of AFP with compound 7 in the dose of 91μg/mouse (complex 1:200) resulted in that the relative amount of AFCincreased by 49% and the total AFC amount increased by 47%; for complexAFP with compound 10 in the dose of 91 μg/mouse (complex 1:200) thecorresponding figures increased by 88% and 79% accordingly; for acomplex AFP with compound 14 in the dose of 91 μg/mouse (complex 1:200)the corresponding figures increased by 55% and 53% accordingly; for acomplex AFP with compound 15 in the dose of 91 μg/mouse (complex 1:200)the corresponding increased by 50% and 48%, respectively. The increaseis calculated as a comparison with the amount of cells in animalsimmunised only with sheep erythrocytes.

All title compounds in the form of complexes with AFP exhibit theimmunostimulating effect in vivo in lower concentrations than thesecompounds alone. It is suggested, taht AFP reduces the critical micelleconcentration (CMC) of the title compounds on account of highlycooperative specific interactions in complexes comprising AFP and aligand. So, in the experiments performed by the inventor, compounds 1,2a, and 3 in a complex with AFP displayed an immunostimulating effect invivo at a dose of 45.5 μg/mouse and compounds 7, 10, 14, and 15—at adose of 91 μg/mouse.

In the experiments disclosed in the present application, the inventorhas used C57B1/6 mice, which are low reactive to sheep red blood cells(SRBC). It possible, that complexes title compounds 1-4, 1a-4a, and 5-20with AFP would display immunostimulating effect at more low amounts, ifmice, which are high reactive to SRBC, have been used. The presentinventor has shown that complex compound 5 (N-AAP) with AFP displaysimmunostimulating effect at 45.5 μg/mouse, in experiments with CBA mice(See the International Application No PCT/EP99/04201).

AFP was isolated from human cord blood by immunoaffinity chromatographyon monoclonal antibodies against AFP immobilised on Sepharose®immunoaffinity chromatography on polyclonal antibodies to the proteinsof normal human blood and gel-filtration on Sephacryl S-200®. The AFPpreparation thus obtained exhibited a purity above 99% and did notcontain low molecular weight impurities and retained completely itsbiological activity.

Rat AFP was isolated from neonatal rat serum. Monospecific anti-ratserum alfa-fetoprotein IgG was coupled to cyanogen bromide-activatedSepharose® 4B (4.5 mg/ml packed volume of gel) to yield animmunoaffinity matrix. Acidic elution conditions were as describedpreviously (Calvo M., et al., J.Chromatogr. Vol.328, p. 392-395, 1985).

Other sources of AFP may be purified and/or modified AFP from othermammals, for example from genetically modified mammals, or from cellcultures. Preferably, the AFP is biotechnologically manufactured using acell culture of genetically modified cells expressing human AFP. Withknowledge of the nucleotide sequence coding for human AFP, this can beinserted in a host, together with necessary promoters and other sequenceinformation, for example sequences influencing the extracelluarexpression of AFP. The AFP is collected from the cell culture andpurified by chromatography and may be further purified bygel-filtration. In any case the production method must involve steps,which guarantee that the final product is free from pyrogens andpossible viral or bacterial contaminants. Suitable production methodscan for example be found in the field of interferon production.

The novel retinoids according to the present invention exhibit goodsolubility in water due to the presence of the phosphate groups in themolecules. The water solubility opens many opportunities for obtainingvarious medical forms of the preparations possessing suitable propertiesfor transdermal and/or oral absorption.

The compounds according to the invention can be used as such or ascomponents in pharmaceutical compositions. The compounds can be givensystemically or locally, for example topically. Suitable modes ofadministration of the compounds include intravenous administration,intraperitoneal administration, as well as oral, rectal and transdermaladministration. The intended mode of administration is naturally takeninto account in the preparation of the final pharmaceutical composition.Normal pharmaceutical adjuvants can naturally be used and the compoundscan be made available in the form of injectable solutions, ointments,capsules or tablets, in the form of transdermal patches or suppositoriesaccording to the intended use and/or mode of administration.

The therapeutic effective doses for intravenous administration of thetitle compounds are in intervals:

Compounds 1, 1a, 5, 9, 13, 17: from 5 mg/kg to 10 mg/kg

Compounds 2, 2a, 3, 3a, 6, 7, 10, 11, 14, 15, 18, 19: from 5 mg/kg to 20mg/kg

Compounds 4, 4a, 8, 12, 16, 20: from 5 mg/kg to 30 mg/kg

The therapeutic effective doses for intravenous administration complexesof AFP with title compounds are in intervals:

Compounds 1, 1a, 5, 9, 13, 17: from 2 mg/kg to 10 mg/kg

AFP: from 0.2 mg/kg to 1 mg/kg

Compounds 2, 2a, 3, 3a, 6, 7, 10, 11, 14, 15, 18, 19: from 2 mg/kg to 20mg/kg

AFP: from 0.2 mg/kg to 2 mg/kg

Compounds 4, 4a, 8, 12, 16, 20: from 2 mg/kg to 30 mg/kg

AFP: from 0.2 mg/kg to 3 mg/kg

EXAMPLES Materials and Methods

L-Serine, L-threonine, L-tyrosine, and arachidonic, eicosapentaenoic,linoleic, docosahexaenoic and retinoic acids were purchased from SigmaChemical Co. Ethanolamine, N,N′-dicyclohexylcarbodiimide, and2-cyanoethyl phosphate barium salt were obtained from Aldrich ChemicalCo. Tetrahydrofuran, acetonitrile and pyridine were dried by heating,under reflux, with CaH₂ for 3-5 h; these solvents were then distilled atatmospheric pressure and stored over molecular sieves (no. 4A).

The barium salt of 2-cyanoethyl phosphate was converted into thepyridinium salt by passage through a column of Dowex-50 resin(pyridinium form). The eluate was evaporated and the salt was dried byrepeated evaporation of added portions of dry pyridine.

¹H-NMR spectra at 200 MHz were obtained with a Brucker spectrometerAC-200; tetramethylsilane was used as an internal standard.

Merck silica gel 60 pre-coated plates, which were developed in solventsystem A [benzene-dioxan-acetic acid (25:5:1 v/v/v)] and solvent systemB [chloroform-methanol-NH₃aq (9:7:2 v/v/v)] were used for thin-layerchromatography (TLC). Detection of the compounds on TLC plates was byspraying with 10% sulphuric acid in methanol or with molybdate spray.Flash chromatography was performed on silica gel 60 (230-400 mesh).

Example 1

Synthesis of theN-(cis-5,8,11,14-eicosatetraenoyl)-O-phospho-2-aminoethanol(N-arachidonoyl-O-phospho-2-aminoethanol) (5)

Arachidonic acid (152 mg, 0.5 mmol) and triethylamine (52 mg, 0.51 mmol)were dissolved in 3 ml of dry acetonitrile and chilled to −15° C., and70 mg (0.51 mmol) of butyl chloroformate was added. After 30 min, themixture free of the precipitated triethylamine hydrochloride waspipetted in a solution of 2-aminoethanol (61 mg, 1 mmol) in 1 ml ofmethanol, stirring was continued for 15 min at −15° C., then the mixtureobtained was allowed to warm to room temperature. After 2 h, 0.5 M HClwas added, and the mixture was extracted with ether (20 ml). The extractwas washed with water, then dried with Na₂SO₄, and evaporated underreduced pressure. The residue was dissolved in 2 ml of chloroform andpurified by column (2×2 cm) chromatography on aluminum oxide (basic,Brockmann II). Elution of the column with chloroform-methanol (9:1 v/v)and evaporation of the appropriate fractions gave 165 mg (95%) ofdesired N-arachidonoylaminoethan-2-ol as oil: TLC (system A) R_(f) 0.4.

A solution of pyridinium cyanoethylphosphate (2 mmol) in anhydrouspyridine (3 ml) was added to dry N-acylaminoethan-2-ol.N,N′-Dicyclohexylcarbodiimide (413 mg, 2 mmol) was then added and themixture was stirred at room temperature. After 20 h, the mixture wascooled to 0° C., water (0.5 ml) was added and, after stirring for 30 minat room temperature, the precipitated N,N′-dicyclohexylurea wasseparated by filtration. The filtrate was evaporated under reducedpressure and the residue obtained was fractionated by short columnchromatography on silica gel. The desired phosphorylatedN-acylaminoalcohol was eluted from column with chloroform-methanol(70-60:30-40, v/v). The composition of the eluates was controlled by TLCon Silica gel 60 plates (system B) using a molybdate spray for detectingthe spots. The appropriate fractions were combined, evaporated todryness in vacuo and residue was dissolved in 1 ml tetrahydrofuran. Thatsolution was added , dropwise over a period of 5 min, to a cooled(ice-bath), stirred 1.5 M NaOHaq (4 ml). After a further 25 min, themixture was acidified with 1 N HCl to pH 2-3 and extracted withchloroform-methanol (2:1, v/v). The extract was washed withmethanol-water (10:9, v/v), concentrated in vacuo and applied to acolumn of silica gel. The desired product was eluted from column withchloroform-methanol (30-20:70-80, v/v), the fractions containing puresubstance stained on the TLC plates with molybdate spray were combinedand evaporated to dryness to give 88 mg (41%) of (5): R_(f) 0.10-0.15(system B); ¹H-NMR (CD₃SOCD₃, 200 MHz) δ0.9-1.0 (t, 3H, ω-CH₃); 1.3 (s,8H, 4CH₂); 2.0-2.4 (m, 6H, 2CH₂CH═CH and CH₂CO); 2.7-2.9 (br s, 6H,3HC═CHCH₂CH═CH); 3.4-3.5 (br s, 2H, CH₂NH); 3.9-4.0 (br s, 2H, CH₂OP);5.2-5.4 (br s, 8H, 4HC═CH); 8.2-8.4 (m, 3H, NH and 2POH).

Example 2

Synthesis of theN-(cis-4,7,10,13,16,19-docosahexaenoyl)-O-phospho-2-aminoethanol (9)

This compound was prepared as described above for (5), using 0.5 mmol(164 mg) of cis-4,7,10,13,16,19-docosahexaenoic acid; yield 99 mg (44%);R_(f) 0.10-0.15 (system B); ¹H NMR (CD₃SOCD₃, 200 MHz) δ0.9-1.0 (t, 3H,ω-CH₃); 2.0-2.1 (t, 2H, CH₂CO); 2.2-2.4 (m, 4H, 2CH₂CH═CH); 2.7-2.9 (brs, 10H, 5HC═CHCH₂CH═CH); 3.4-3.5 (br s, 2H, CH₂NH); 3.9-4.0 (br s, 2H,CH₂OP); 5.2-5.4 (br s, 12H, 6HC═CH); 8.2-8.4 (m, 3H, NH and 2POH).

Example 3

Synthesis of the N-(all-trans-Retinoyl)-O-phospho-2-aminoethanol (1)

all-trans-Retinoic acid (150 mg, 0.5 mmol) and triethylamine (52 mg,0.51 mmol) were dissolved in 1 ml of dry tetrahydrofuran, whereupon dryacetonitrile (2 ml) was added, and the mixture was chilled to −15° C.All further procedures were carried out as described above for (5);yield 59 mg,(28%); R_(f) 0.10-0.15 (system B); λ_(max) (ethanol) 345 nm;¹H-NMR (CD₃SOCD₃, 200 MHz) δ1.0 (s, 6H, 3CH₂, ring); 1.4-2.4 (m, 15H,5CH₃); 3.4-3.5 (br s, 2H, CH₂NH); 3.9-4.0 (br s, 2H, CH₂OP); 5.8-7.0 (m,6H, 6HC═C); 8.2-8.4 (m, 3H, NH and 2POH)

Example 4

Synthesis of the N-(cis-5,8,11,14-eicosatetraenoyl)-O-phospho-L-serine(N-arachidonoyl-O-phospho-L-serine) (6)

Arachidonic acid (152 g, 0.5 mmol) and triethylamine (52 mg, 0.51 mmol)were dissolved in 3 ml of dry acetonitrile and chilled to −15° C., and70 mg (0.51 mmol) of butyl chloroformate was added. After 30 min, themixture free of the precipitated triethylamine hydrochloride waspipetted in a solution of L-serine methyl ester hydrochloride (156 mg, 1mmol) and 0.14 ml of triethylamine in 1 ml of methanol, stirring wascontinued for 15 min at −15° C., then that mixture was allowed to warmto room temperature. After 2 h, 0.5 M HCl was added, and the mixture wasextracted with ether (20 ml). The extract was washed with water, thendried with Na₂SO₄, and evaporated in vacuo. The residue was dissolved in2 ml of chloroform and purified by column (2×2 cm) chromatography onaluminum oxide (basic, Brockmann II). Elution of the column withchloroform-methanol (9:1 v/v) and evaporation of the appropriatefractions gave 186 mg (95%) of desired N-arachidonoyl-L-serine methylester as oil: TLC (system A) R_(f) 0.5.

A solution of pyridinium cyanoethylphosphate (2 mmol) in anhydrouspyridine (3 ml) was added to dry N-arachidonoyl-L-serine methyl ester.N,N′-Dicyclohexylcarbodiimide (413 mg, 2 mmol) was then added and themixture was stirred at room temperature. After 20 h, the mixture wascooled to 0° C., water (0.5 ml) was added and, after stirring for 30 minat room temperature, the precipitated N,N′-dicyclohexylurea wasseparated by filtration. The filtrate was evaporated in vacuo and theresidue obtained was fractionated by short column chromatography onsilica gel. The desired phosphorylated N-arachidonoyl-L-serine methylester was eluted from column with chloroform-methanol (30-40:70-60,v/v). The composition of the eluates was controlled by TLC on Silica gel60 plates (system B) using a molybdate spray for detecting the spots.The appropriate fractions were combined, evaporated to dryness in vacuoand residue was dissolved in 1 ml tetrahydrofuran. The solution obtainedwas added, dropwise over a period of 5 min, to a cooled (ice-bath),stirred 1.5 M NaOHaq (4 ml). After a further 25 min, the mixture wasacidified with 1 N HCl to pH 2-3 and extracted with chloroform-methanol(70-60:30-40, v/v). The extract was washed with methanol-water (10:9,v/v), concentrated in vacuo and applied to a column of silica gel. Thedesired product was eluted from column with chloroform-methanol(30-20:70-80, v/v), the fractions containing pure substance stained onthe TLC plates with molybdate spray were combined and evaporated todryness to give 82 mg (35%) of (6): R_(f) 0.05-0.10 (system B); ¹H-NMR(CD₃SOCD₃, 200 MHz) δ0.9-1.0 (t, 3H, ω-CH₃); 1,3 (s, 8H, 4CH₂); 2.0-2.4(m, 6H, 2CH₂CH═CH and CH₂CO); 2.7-2.9 (br s, 6H, 3HC═CHCH₂CH═CH);3.9-4.0 br s, 2H, CH₂OP); 4.3-4.4 (m, 1H, NHCHCO); 5.2-5.4 (br s, 8H,4HC═CH); 8.2-8.4 (m, 3H, NH and 2POH)

Example 5

Synthesis of theN-(cis-5,8,11,14-eicosatetraenoyl)-O-phospho-L-threonine(N-arachidonoyl-O-phospho-L-threonine) (7)

This compound was prepared as described above for (6) using 1 mmol (170mg) of L-threonine methyl ester hydrochloride; yield 97 mg, (40%); R_(f)0.05-0.10 (system B); ¹H-NMR (CD₃SOCD₃, 200 MHz) δ0.9-1.0 (t, 3H,ω-CH₃); 1.2-1.4 (m, 11H, 4CH₂ and CH₃CHOP); 2.0-2.4 (m, 6H, 2CH₂CH═CHand CH₂CO); 2.7-2.9 (br s, 6H, 3HC═CHCH₂CH═CH); 4.1-4.3 (m, 2H, 2CH);5.2-5.4 (br s, 8H, 4HC═CH); 8.2-8.4 (m, 3H, NH and 2POH)

Example 6

Synthesis of theN-(cis-4,7,10,13,16,19-docosahexaenoyl)-O-phospho-L-serine (10)

This compound was prepared as described above for (6) using 0.5 mmol(164 mg) of cis-4,7,10,13,16,19-docosahexaenoic acid; yield 91 mg (37%);R_(f) 0.05-0.10 (system B); ¹H-NMR (CD₃SOCD₃, 200 MHz) δ0.9-1.0 (t, 3H,ω-CH₃); 2.0-2.1 (t, 2H, CH₂CO); 2.2-2.4 (m, 4H, 2CH₂CH═CH); 2.7-2.9 (brs, 10H, 5HC═CHCH₂CH═CH); 3.9-4.0 (br s, 2H, CH₂OP); 4.3-4.4 (m, 1H,NHCHCO); 5.2-5.4 (br s, 12H, 6HC═CH); 8.2-8.4 (m, 3H, NH and 2POH)

Example 7

Synthesis of theN-(cis-4,7,10,13,16,19-docosahexaenoyl)-O-phospho-L-threonine (11)

This compound was prepared as described above for (6) using 0.5 mmol(164 mg) of cis-4,7,10,13,16,19-docosahexaenoic acid and 1 mmol (170 mg)of L-threonine methyl ester hydrochloride; yield 109 mg, (43%); R_(f)0.05-0.10 (system B); ¹H-NMR (CD₃SOCD₃, 200 MHz) δ0.9-1.0 (t, 3H,ω-CH₃); 1.2-1.4 (3H, d, CH₃CHOP); 2.0-2.1 (t, 2H, CH₂CO); 2.2-2.4 (m,4H, 2CH₂CH═CH); 2.7-2.9 (br s, 10H, 5HC═CHCH₂CH═CH); 4.1-4.3 (m, 2H,2CH); 5.2-5.4 (br s, 12H, 6HC═CH); 8.2-8.4 (m, 3H, NH and 2POH)

Example 8

Synthesis of the N-(all-trans-Retinoyl)-O-phospho-L-serine (2)

all-trans-Retinoic acid (150 mg, 0.5 mmol) and triethylamine (52 mg,0.51 mmol) were dissolved in 1 ml of dry tetrahydrofuran, then dryacetonitrile (2 ml) was added, and the mixture was chilled to −15° C.All further procedures were carried out as described for (6); yield 56mg, (24%); R_(f) 0.05-0.10 (system B); λ_(max) (ethanol) 345 nm; ¹H-NMR(CD₃SOCD₃, 200 MHz) δ1.0 (s, 6H, 3CH₂, ring); 1.4-2.4 (m, 15H, 5CH₃);3.9-4.0 (br s, 2H, CH₂OP); 4.3-4.4 (m, 1H, NHCHCO); 5.8-7.0 (m, 6H,6HC═C); 8.2-8.4 (m, 3H, NH and 2POH)

Example 9

Synthesis of the N-(all-trans-Retinoyl)-O-phospho-L-threonine (3)

all-trans-Retinoic acid (150 mg, 0.5 mmol) and triethylamine (52 mg,0.51 mmol) were dissolved in 1 ml of dry tetrahydrofuran, then dryacetonitrile (2 ml) was added, and the mixture was chilled to −15° C.All further procedures were carried out as described for (6) using 1mmol (170 mg) of L-threonine methyl ester hydrochloride; yield 65 mg,(27%); R_(f) 0.05-0.10 (system B); λ_(max) (ethanol) 345 nm; ¹H-NMR(CD₃SOCD₃, 200 MHz) δ1.0 (s, 6H, 3CH₂, ring); 1.2-1.4 (d, 3H, CH₃CHOP);1.4-2.4 (m, 15H, 5CH₃); 4.1-4.3 (m, 2H, 2CH); 5.8-7.0 (m, 6H, 6HC═C);8.2-8.4 (m, 3H, NH and 2POH)

Example 10

Synthesis of the N-(cis-5,8,11,14-eicosatetraenoyl)-O-phospho-L-tyrosine(N-arachidonoyl-O-phospho-L-tyrosine) (8)

Arachidonic acid (152 g, 0.5 mmol) and triethylamine (52 mg, 0.51 mmol)were dissolved in 3 ml of dry acetonitrile and chilled to −15° C., and70 mg (0.51 mmol) of butyl chloroformate was added. After 30 min, themixture free of the precipitated triethylamine hydrochloride waspipetted in a solution of tyrosine methyl ester hydrochloride (232 mg, 1mmol) and 0.14 ml of triethylamine in 1 ml of methanol, stirring wascontinued for 15 min at −15° C., then the mixture obtained was allowedto warm to room temperature. After 2 h, 0.5 M HCl was added, and themixture was extracted with ether (20 ml). The extract was washed withwater, then dried with Na₂SO₄, and evaporated in vacuo. The residue wasdissolved in 2 ml of chloroform and purified by column (2×2 cm)chromatography on aluminum oxide (basic, Brockmann II). Elution of thecolumn with chloroform-methanol (9:1 v/v) and evaporation of theappropriate fractions gave 222 mg (95%) of desiredN-arachidonoyl-L-tyrosine methyl ester as oil: TLC (system A) R_(f) 0.6.

A solution of pyridinium cyanoethylphosphate (2 mmol) in anhydrousacetonitrile (3 ml) was added to dry N-arachidonoyl-L-tyrosine methylester. N,N′-Dicyclohexylcarbodiimide (413 mg, 2 mmol) was then added andthe mixture was stirred at room temperature. After 5 days, the mixturewas cooled to 0° C., water (0.5ml) was added and, after stirring for 30min at room temperature, the precipitated N,N′-dicyclohexylurea wasseparated by filtration. The filtrate was evaporated in vacuo and theresidue obtained was fractionated by short column chromatography onsilica gel. The desired phosphorylated N-acyl-L-tyrosine methyl esterwas eluted from column with chloroform-methanol (70-60:30-40, v/v). Thecomposition of the eluates was controlled by TLC on Silica gel 60 plates(system B) using a molybdate spray for detecting the spots. Theappropriate fractions were combined, evaporated to dryness in vacuo andresidue was dissolved in 1 ml tetrahydrofuran. The solution obtained wasadded, dropwise over a period of 5 min, to a cooled (ice-bath), stirred1.5 M NaOHaq (4 ml). After a further 25 min, the mixture was acidifiedwith 1 N HCl to pH 2-3 and extracted with chloroform-methanol (2:1,v/v). The extract was washed with methanol-water (10:9, v/v),concentrated in vacuo and applied to a column of silica gel. The desiredproducts was eluted from column with chloroform-methanol (30-20:70-80,v/v), the fractions containing pure substance stained on the TLC plateswith molybdate spray were combined and evaporated to dryness to give 63mg (23%) of (8): R_(f) 0.05-0.10 (system B); ¹H-NMR (CD₃SOCD₃, 200 MHz)δ0.9-1.0 (t, 3H, ω-CH₃); 1.3 (s, 8H, 4CH₂); 2.0-2.4 (m, 6H, 2CH₂CH═CHand CH₂CO); 2.7-2.9 (br s, 6H, 3HC═CHCH₂CH═CH); 2.9-3.1 (m, 2H, CH₂Ar);4.3-4.4 (m, 1H, NHCHCO); 5.2-5.4 (br s, 8H, 4HC═CH); 7.0-7.2 (q, 4H,Ar); 8.2-8.4 (m, 3H, NH and 2POH)

Example 11

Synthesis of theN-(cis-4,7,10,13,16,19-docosahexaenoyl)-O-phospho-L-tyrosine (12)

This compound was prepared as described above for (8) using 0.5 mmol(164 mg) of cis-4,7,10,13,16,19-docosahexaenoic acid; yield 74 mg,(26%); R_(f) 0.05-0.10 (system B); ¹H-NMR (CD₃SOCD₃, 200 MHz) δ0.9-1.0(t, 3H, ω-CH₃); 2.0-2.1 (t, 2H, CH₂CO); 2.2-2.4 (m, 4H, 2CH₂CH═CH);2.7-2.9 (br s, 10H, 5HC═CHCH₂CH═CH); 2.9-3.1 (m, 2H, CH₂Ar); 4.3-4.4 (m,1H, NHCHCO); 5.2-5.4 (br s, 12H, 6HC═CH); 7.0-7.2 (q, 4H, Ar); 8.2-8.4(m, 3H, NH and 2POH)

Example 12

Synthesis of the N-(all-trans-Retinoyl)-O-phospho-L-tyrosine (4)

all-trans-Retinoic acid (150 mg, 0.5 mmol) and triethylamine (52 mg,0.51 mmol) were dissolved in 1 ml of dry tetrahydrofuran, then dryacetonitrile (2 ml) was added, and the mixture was chilled to −15° C.All further procedures were carried out as described for (8) using 1mmol (232 mg) of L-tyrosine methyl ester hydrochloride; yield 52 mg,(19%); R_(f) 0.05-0.10 (system B); λ_(max) (ethanol) 345 nm; ¹H-NMR(CD₃SOCD₃, 200 MHz) δ1.0 (s, 6H, 3CH₂, ring); 1.4-2.4 (m, 15H, 5CH₃);2.9-3.1 (m, 2H, CH₂Ar); 4.3-4.4 (m, 1H, NHCHCO); 5.8-7.0 (m, 6H, 6HC═C);7.0-7.2 (q, 4H, Ar); 8.2-8.4 (m, 3H, NH and 2POH)

Example 13

Synthesis of the N-(13cis-Retinoyl)-O-phospho-2-aminoethanol (1a)

This compound was prepared as described above for (1) using 0.5 mmol(150 mg) of 13-cis-retinoic acid; yield 63 mg, (30%); R_(f) 0.10-0.15(system B); λ_(max) (ethanol) 347 nm; ¹H-NMR (CD₃SOCD₃, 200 MHz) δ1.0(s, 6H, 3CH₂, ring); 1.1-2.2 (m, 15H, 5CH₃); 3.3-3.4 (br s, 2H, CH₂NH);3.8-3.9 (br s, 2H, CH₂ OP); 5.8-7.0 (m, 6H, 6HC═C); 8.2-8.4 (m, 3H, NHand 2POH)

Example 14

Synthesis of the N-(13-cis-Retinoyl)-O-phospho-L-serine (2a)

This compound was prepared as described above for (2) using 0.5 mmol(150 mg) of 13-cis-Retinoic acid; yield 54 mg, (23%); R_(f) 0.05-0.10(system B); λ_(max) (ethanol) 347 nm; ¹H-NMR (CD₃SOCD₃, 200 MHz) δ1.0(s, 6H, 3CH₂, ring); 1.1-2.2 (m, 15H, 5CH₃); 3.8-3.9 (br s, 2H, CH₂OP);4.2-4.3 (m, 1H, NH CH CO); 5.8-7.0 (m, 6H, 6HC═C); 8.2-8.4 (m, 3H, NHand 2POH)

Example 15

Synthesis of the N-(13-cis-Retinoyl)-O-phospho-L-threonine (3a)

This compound was prepared as described above for (3) using 0.5 mmol(150 mg) of 13-cis-Retinoic acid; yield 70 mg, (29%); R_(f) 0.05-0.10(system B); λ_(max) (ethanol) 347 nm; ¹H-NMR (CD₃SOCD₃, 200 MHz) δ1.0(s, 6H, 3CH₂, ring); 1.1-2.2 (m, 18H, 6CH₃); 4.1-4.3 (m, 2H, 2CH);5.8-7.0 (m, 6H, 6HC═C); 8.2-8.4 (m, 3H, NH and 2POH)

Example 16

Synthesis of the N-(13-cis-Retinoyl)-O-phospho-L-tyrosine (4a)

This compound was prepared as described above for (4) using 0.5 mmol(150 mg) of 13-cis-Retinoic acid; yield 54 mg, (20%); R_(f) 0.05-0.10(system B); λ_(max) (ethanol) 347 nm; ¹H-NMR (CD₃SOCD₃, 200 MHz) δ1.0(s, 6H, 3CH₂, ring); 1.1-2.2 (m, 15H, 5CH₃); 2.9-3.1 (m, 2H, CH₂Ar);4.2-4.3 (m, 1H, NH CH CO); 5.8-7.0 (m, 6H, 6HC═C); 7.1 (s, 4H, Ar);8.2-8.4 (m, 3H, NH and 2POH)

Example 17

Synthesis of theN-(cis-5,8,11,14,17-eicosapentaenoyl)-O-phospho-2-aminoethanol (13)

This compound was prepared as described above for (5) using 0.5 mmol(151 mg) of cis-5,8,11,14,17-eicosapentaenoic acid; yield 98 mg, (46%);R_(f) 0.10-0.15 (system B); ¹H-NMR (CD₃SOCD₃, 200 MHz) δ0.9-1.0 (t, 3H,ω-CH₃); 1.5-1.6 (t, 2H, CH₂); 2.0-2.2 (m, 6H, CH₂CO and 2CH₂CH═CH);2.7-2.9 (br s, 8H, 4HC═CH—CH₂—CH═CH); 3.2-3.3 (br s, 2H, CH₂NH); 3.8-3.9(br s, 2H, CH₂OP); 5.2-5.4 (br s, 10H, 5 HC═CH); 8.2-8.4 (m, 3H, NH and2POH)

Example 18

Synthesis of theN-(cis-5,8,11,14,17-eicosapentaenoyl)-O-phospho-L-serine (14)

This compound was prepared as described above for (6) using 0.5 mmol(151 mg) of cis-5,8,11,14,17-eicosapentaenoic acid; yield 84 mg, (36%);R_(f) 0.05-0.10 (system B); ¹H-NMR (CD₃SOCD₃, 200 MHz) δ0.9-1.0 (t, 3H,ω-CH₃); 1.5-1.6 (t, 2H, CH₂); 2.0-2.2 (m, 6H, CH₂CO and 2 CH₂CH═CH);2.7-2.9 (br s, 8H, 4HC═CH—CH₂—CH═CH); 3.9-4.0 (br s, 2H, CH₂OP); 4.34.4(m, 1H, NH CHCO); 5.2-5.4 (br s, 10H, 5 HC═CH); 8.2-8.4 (m, 3H, NH and2POH)

Example 19

Synthesis of theN-(cis-5,8,11,14,17-eicosapentaenoyl)-O-phospho-L-threonine (15)

This compound was prepared as described above for (6), using 0.5 mmol(151 mg) of cis-5,8,11,14,17-eicosapentaenoic acid and 1 mmol (170 mg)of L-threonine methyl ester hydrochloride; yield 99 mg, (41%); R_(f)0.05-0.10 (system B); ¹H-NMR (CD₃SOCD₃, 200 MHz) δ0.9-1.0 (t, 3H,ω-CH₃); 1.2-1.4 (d, 3H, CH₃CHOP); 1.5-1.6 (t, 2H, CH₂); 2.0-2.2 (m, 6H,CH₂CO and 2CH₂CH═CH); 2.7-2.9 (br s, 8H, 4HC═CH—CH₂—CH═CH); 4.1-4.3 (m,2H, 2CH); 5.2-5.4 (br s, 10H, 5HC═CH); 8.2-8.4 (m, 3H, NH and 2POH)

Example 20

Synthesis of theN-(cis-5,8,11,14,17-eicosapentaenoyl)-O-phospho-L-tyrosine (16)

This compound was prepared as described above for (8), using 0.5 mmol(151 mg) of cis-5,8,11,14,17-eicosapentaenoic acid; yield 65 mg, (24%);R_(f) 0.05-0.10 (system B); ¹H-NMR (CD₃SOCD₃, 200 MHz) δ0.9-1.0 (t, 3H,ω-CH₃); 1.5-1.6 (t, 2H, CH₂); 2.0-2.2 (m, 6H, CH₂CO and 2CH₂CH═CH);2.7-3.0 (m, 10H, 4HC═CH—CH₂—CH═CH and CH₂Ar); 4.3-4.4 (m, 1H, NHCHCO);5.2-5.4 (br s, 10H, 5HC═CH); 7.1 (s, 4H, Ar); 8.2-8.4 (m, 3H, NH and2POH)

Example 21

Synthesis of theN-(cis-9,12,15-octadecatrienoyl)-O-phospho-2-aminoethanol(N-linolenoyl-O-phospho-2-aminoethanol) (17)

This compound was prepared as described above for (5), using 0.5 mmol(139 mg) of cis-9,12,15-octadecatrienoic acid; yield 80 mg, (40%); R_(f)0.10-0.15 (system B); ¹H-NMR (CD₃SOCD₃, 200 MHz) δ0.9-1.0 (t, 8H,ω-CH₃); 1.3(s, 8H, 4CH₂); 1.4-1.5(br, s, 2H, CH₂CH₂CO); 2.0-2.2 (m, 6H,CH₂CO and 2CH₂CH═CH); 2.7-2.9 (br s, 4H, 2HC═CH—CH₂—CH═CH); 3.1-3.2 (brs, 2H, CH₂NH); 3.7-3.8 (br s, 2H, CH₂OP); 5.2-5.4 (br s, 6H, 3HC═CH);8.2-8.4 (m, 3H, NH and 2POH).

Example 22

Synthesis of the N-(cis-9,12,15-octadecatrienoyl)-O-phospho-L-serine(N-linolenoyl-O-phospho-L-serine) (18)

This compound was prepared as described above for (6), using 0.5 mmol(139 mg) of cis-9,12,15-octadecatrienoic acid; yield 73 mg (33%); R_(f)0.05-0.10 (system B); ¹H-NMR (CD₃SOCD₃, 200 MHz) δ0.9-1.0 (t, 3H,ω-CH₃); 1.3 (s, 8H, 4CH₂); 1.4-1.5 (br s, 2H, CH₂CH₂CO); 2.0-2.2 (m, 6H,CH₂CO and 2 CH₂CH═CH); 2.7-2.9 (br s, 4H, 2HC═CH—CH₂—CH═CH); 3.9-4.0 (brs, 2H, CH₂OP); 4.3-4.4 (m, 1H, NH CHCO); 5.2-5.4 (br s, 6H, 3 HC═CH);8.2-8.4 (m, 3H, NH and 2POH).

Example 23

Synthesis of the N-(cis-9,12,15-octadecatrienoyl)-O-phospho-L-threonine(N-linolenoyl-O-phospho-L-threonine) (19)

This compound was prepared as described above for (6), using 0.5 mmol(139 mg) of cis-9,12,15-octadecatrienoic acid and 1 mmol (1 70 mg) ofL-threonine methyl ester hydrochloride; yield 85 mg (37%); R_(f)0.05-0.10 (system B); ¹H-NMR (CD₃SOCD₃, 200 MHz) δ0.9-1.0 (t, 3H,ω-CH₃); 1.3-1.5 (m, 13H, 5CH₂ and CH₃CHOP); 2.0-2.2 (m, 6H, CH₂CO and2CH₂CH═CH); 2.7-2.9 (br s, 4H, 2HC═CH—CH₂—CH═CH); 4.1-4.3 (m, 2H, 2CH);5.2-5.4 (br s, 6H, 3HC═CH); 8.2-8.4 (m, 3H, NH and 2POH)

Example 24

Synthesis of the N-(cis-9,12,15-octadecatrienoyl)-O-phospho-L-tyrosine(N-linolenoyl-O-phospho-L-tyrosine) (20)

This compound was prepared as described above for (8), using 0.5 mmol(139 mg) of cis-9,12,15-octadecatrienoic acid; yield 52 mg (20%); R_(f)0.05-0.10 (system B); ¹H-NMR (CD₃SOCD₃, 200 MHz) δ0.9-1.0 (t, 3H,ω-CH₃); 1.3(s, 8H, 4CH₂); 1.4-1.5 (br, s, 2H, CH₂CH₂CO); 2.0-2.2 (m, 6H,CH₂CO and 2CH₂CH═CH); 2.7-2.9 (br s, 4H, 2HC═CH—CH₂—CH═CH );2.9-3.1(m,2H, CH₂Ar); 4.3-4.4 (m, 1H, NHCHCO); 5.2-5.4 (br s, 6H, 3HC═CH); 7,0 (s,4H, Ar); 8.2-8.4 (m, 3H, NH and 2POH).

Example 25

The Influence of Compound 10 on Humoral Immune Response

0.15 ml of compound 10 was administered intravenously to 6 mice femalesof CS7B1/6 line (weight 18-22 g) in doses of 45.5, 91 and 136.5 μg ofcompound 10 per capita for each testing group according to the dose.Simultaneously, a suspension of 5·10⁷ sheep erythrocytes was injectedintraperitoneally (0.2 ml per capita). Mice in the control group wereinjected intravenously with equal volume of saline. The effects ofcompound 10 on humoral immune response were analyzed both by countingthe quantity of AFC in the spleen according to Cunningham (per 10⁶spleen cells and per spleen) and estimating the titers of hemagglutiningantibodies in serum of the animals (Cunningham A. J., Nature, 1965,Vol.207, No 5001. P.1106-1107), while the titers of hemagglutinatingantibodies were evaluated according to litterature (Evans J. et al.,Cell, 1974; Vol 3., p.153-158, Feizi T., Menger E., Transfusion, 1970,Vol. 10, p.33-35,).

The relative amount of AFC on the 5-th day after injection was273.3±20.4 for control animals, 304.9±46.6 for the experimental groupwith compound 10 in the dose of 45.5 μg/mouse, p>0.05, 326.2±65.4 forthe group with compound 10 in the dose of 91 μg/mouse, p>0.05, and651.6±48.2 for the experimental group with compound 10 in the dose of136.5 μg/mouse, p<0.001. The total AFC amount was (31.3±2.5)·10³ for thecontrol group, (30.2±5.7)·10³ for the experimental group receivingcompound 10 in the dose of 45.5 μg/mouse, p>0.05, (34.7±8.5)·10³ for thegroup receiving compound 10 in the dose of 91 μg/mouse, p>0.05, and(81.1±5.8)·10³ for the experimental group receiving compound 10 in thedose of 136.5 μg/mouse, p<0.001. Moreover, compound 10 in the dose of136.5 μg/mouse caused significant increase of hemagglutinin titres inserum of immunised mice, those were 7.0±0.4 for control animals and8.5±0.4 for the group receiving compound 10 in the dose of 136.5μg/mouse, p<0.05 (Table 1).

The immunogenic activity of compound 10 is dose-dependent. Compound 10in the doses of 45.5 and 91 μg/mouse exhibits no immunogenic activity,but at the same time compound 13 in the dose of 136.5 μg/mouse displayedhigh immunostimulating action. Thus, the relative amount of AFC on the5-th day after injection of the compound 10 increased by 138%, and totalAFC amount increased by 159% in comparison to the amount of cells inanimals immunized only with sheep erythrocytes, when hemagglutinintitres in serum of immunised mice increased by 21%.

Example 26

The Influence of Compound 11 on Humoral Immune Response

0.15 ml of compound 11 was administered intravenously to 6 female miceof the C57B1/6 line (weight 18-22 g) in doses of 45.5, 91 and 136.5 μgof compound 11 per capita for each testing group according to the dose.Simultaneously, a suspension of 5·10⁷ sheep erytrocytes was injectedintraperitoneally (0.2 ml per capita). Mice in the control group wereinjected intravenously with an equal volume of saline. The effects ofcompound 11 on humoral immune response was analysed both by counting thequantity of AFC in the spleen according to Cunningham (per 10⁶ spleencells and per spleen) and estimating the titers of hemagglutiningantibodies in serum of the animals.

The relative amount of AFC on the 5-th day after injection was799.3±63.3 for control animals, 676.8±77.1 for the experimental groupreceiving compound 11 in the dose of 45.5 μg/mouse, p>0.05, 896.8±111.8for the group receiving compound 11 in the dose of 91 μg/mouse, p>0.05,and 1336.5±59.0 for the experimental group receiving compound 11 in thedose of 136.5 μg/mouse, p<0.001. The total AFC amount was(101.1±8.6)·10³ for the controls, (85.9±8.3)·10³ for the experimentalgroup receiving compound 11 in the dose of 45.5 μg/mouse, p>0.05,(111.2±11.8)·10³ for the group receiving compound 11 in the dose of 91μg/mouse, p>0.05, and (182.5±7.8)·10³ for the experimental groupreceiving compound 11 in the dose of 136.5 μg/mouse, p<0.001. Moreover,compound 11 in the dose of 136.5 μg/mouse caused significant increase ofhemagglutinin titers in serum of immunized mice, those were 8.3±0.3 forcontrol animals and 9.3±0.2 for group with compound 11 in the dose of136.5 μg/mouse, p<0.02 (Table 2).

The immunogenic activity of compound 11 is dose-dependent. Compound 11in the doses of 45.5 and 91 μg/mouse exhibits no immunogenic activity,but at the same time in the dose of 136.5 μg/mouse the same compound 11displayed marked immunostimulating action. Thus, the relative amount ofAFC on the 5-th day after injection of the compound 11 increased by 67%,and the total AFC amount increased by 80% in comparison with amount ofcells in the animals immunized only with sheep erythrocytes, when thehemagglutinin titers in serum of immunized mice increased by 12%.

Example 27

The Influence of Compound 12 on Humoral Immune Response

0.15 ml of compound 12 was administered intravenously to 6 female miceof the C57B1/6 line (weight 18-22 g) in doses of 45.5, 91 and 136.5 μgof compound 12 per capita for each testing group according to the dose.Simultaneously, a suspension of 5·10⁷ sheep erythrocytes was injectedintraperitoneally (0.2 ml per capita). Mice in the control group wereinjected intravenously with an equal volume of saline. The effects ofcompound 12 on humoral immune response was analyzed both by counting thequantity of AFC in the spleen according to Cunningham (per 10⁶ spleencells and per spleen) and estimating the titers of hemagglutiningantibodies in serum of the animals.

The relative amount of AFC on the 5-th day after injection was291.1±14.3 for the control animals, 263.8±23.6 for the experimentalgroup receiving compound 12 in the dose of 45.5 μg/mouse, p>0.05,307.8±30.9 for the group receiving compound 12 in the dose of 91μg/mouse, p>0.05, and 682.6±33.3 for the experimental group receivingcompound 12 in the dose of 136.5 μg/mouse, p<0.001. The total AFC amountwas (35.0±3.3)·10 for the controls, (31.8±2.8)·10³ for the experimentalgroup receiving compound 12 in the dose of 45.5 μg/mouse, p>0.05,(40.2±2.7)·10³ for the group receiving compound 12 in the dose of 91μg/mouse, p>0.05, and (69.6±6.0)·10³ for the experimental groupreceiving compound 12 in the dose of 136.5 μg/mouse, p<0.001(Table 3).

The immunogenic activity of compound 12 is dose-dependent. Compound 12in the doses of 45.5 and 91 μg/mouse exhibits no immunogenic activity,but at the same time in the dose of 136.5 μg/mouse the same compound 12displayed high immunostimulating action. Thus, the relative amount ofAFC on the 5-th day after injection of the compound 12 increased by134%, and total AFC amount increased 99% in comparison with amount ofcells in the animals immunized only with sheep erythrocytes.

Example 28

The Influence of Compound 8 on Humoral Immune Response

0.15 ml of compound 8 was administered intravenously to 6 female mice ofthe C57B1/6 line (weight 18-22 g) in doses of 45.5, 91 and 136.5 μg ofcompound 8 per capita for each testing group according to the dose.Simultaneously, a suspension of 5·10⁷ sheep erythrocytes was injectedintraperitoneally (0.2 ml per capita). Mice of the control group wereinjected intravenously with an equal volume of saline. The effects ofcompound 8 on humoral immune response were analyzed both by counting thequantity of AFC in the spleen according to Cunningham (per 10⁶ spleencells and per spleen) and estimating the titers of hemagglutiningantibodies in serum of the animals.

The relative amount of AFC on the 5-th day after injection was501.7±21.0 for the control animals, 449.1±37.4 for the experimentalgroup receiving compound 8 in the dose of 45.5 μg/mouse, p>0.05,592.2±41.6 for the group receiving compound 8 in the dose of 91μg/mouse, p>0.05, and 657.8±24.0 for the experimental group receivingcompound 8 in the dose of 136.5 μg/mouse, p<0.001. The total AFC amountwas (46.5±2.4)·10³ for control group, (44.4±4.3)·10³ for theexperimental group receiving compound 8 in the dose of 45.5 μg/mouse,p>0.05, (50.9±4.5)·10³ for the group receiving compound 8 in the dose of91 μg/mouse, p>0.05, and (88.8±8.0)·10³ for the experimental groupreceiving compound 8 in the dose of 136.5 μg/mouse, p<0.001 (Table 4).

The immunogenic activity of compound 8 is dose-dependent. Compound 8 inthe doses of 45.5 and 91 μg/mouse exhibits no immunogenic activity, butat the same time in the dose of 136.5 μg/mouse the compound 8 displayedmarked immunostimulating action. Thus, the relative amount of AFC on the5-th day after injection of the compound 8 increased by 31%, and totalAFC amount increased by 91% in comparison with the amount of cells inthe animals immunised only with sheep erythrocytes.

Example 29

The Influence of Compound 3 on Humoral Immune Response

0.15 ml of compound 3 was administered intravenously to 6 female mice ofthe C57B1/6 line (weight 18-22 g) in doses of 45.5, 91 and 136.5 μg ofcompound 3 per capita for each testing group according to the dose.Simultaneously, a suspension of 5·10⁷ sheep erytrocytes was injectedintraperitoneally (0.2 ml per capita). Mice of the control group wereinjected intravenously with equal volume of saline. The effects ofcompound 3 on humoral immune response were analysed both by counting thequantity of AFC in the spleen according to Cunningham (per 10⁶ spleencells and per spleen) and estimating the titers of hemagglutiningantibodies in serum of the animals.

The relative amount of AFC on the 5-th day after injection was407.4±10.7 for control animals; 472.2±52.4 for the experimental groupreceiving compound 3 in the dose of 45.5 μg/mouse, p>0.05; 827.7±34.6for the group receiving compound 3 in the dose of 91 μg/mouse, p<0.001,and 961.3±56.5 for the experimental group receiving compound 3 in thedose of 136.5 μg/mouse, p<0.001. The total AFC amount was (40.5±2.9)·10³for the control group; (44.6±3.9)·10³ for the experimental groupreceiving compound 3 in the dose of 45.5 μg/mouse, p>0.05,(60.3±4.1)·10³ for the group receiving compound 3 in the dose of 91μg/mouse, p<0.01, and (107.0±8.5)·10³ for the experimental groupreceiving compound 3 in the dose of 136.5 μg/mouse, p<0.001. Moreover,compound 3 in doses of 91 and 136.5 μg/mouse caused significant increaseof the hemagglutinin titers in serum of immunised mice, those were6.3±0.3 for control animals, 8.0±0.3 for the group receiving compound 3in the dose of 91 μg/mouse, p<0.01, and 8.7±0.3 for the group receivingcompound 3 in the dose of 136.5 μg/mouse, p<0.001 (Table 5).

The immunogenic activity of compound 3 is dose-dependent. Compound 3 inthe dose of 45.5 μg/mouse exhibits no immunogenic activity, but at thesame time in doses of 91 and 136.5 μg/mouse compound 3 displayed highimmunostimulating action. Thus, the relative amount of AFC on the 5-thday after injection of the compound 3 increased by 103 and 136%, andtotal AFC amount increased by 49% and 164% accordingly in comparisonwith the amount of cells in the animals immunized only with sheeperythrocytes, when the hemagglutinin titer in serum of immunized miceincreased by 27% and 38%.

Example 30

The Influence of Compound 4 on Humoral Immune Response

0.15 ml of compound 4 was administered intravenously to 6 female mice ofthe C57B1/6 line (weight 18-22 g) in a doses of 45.5, 91 and 136.5 μg ofcompound 4 per capita for each testing group according to the dose.Simultaneously, a suspension of 5·10⁷ sheep erythrocytes was injectedintraperitoneally (0.2 ml per capita). Mice of the control group wereinjected intravenously with an equal volume of saline. The effects ofcompound 4 on humoral immune response were analyzed both by counting thequantity of AFC in the spleen according to Cunningham (per 10⁶ spleencells and per spleen) and estimating the titers of hemagglutiningantibodies in serum of the animals.

The relative amount of AFC on the 5-th day after injection was406.4±16.8 for the control animals; 405.0±47.9 for the experimentalgroup receiving compound 4 in a dose of 45.5 μg/mouse, p>0.05;692.9±44.8 for the group receiving compound 4 in a dose of 91 μg/mouse,p<0.001, and 1354.3±99.9 for the experimental group receiving compound 4in a dose of 136.5 μg/mouse, p<0.001. The total AFC amount was(60.4±3.9)·10³ for the control group, (55.0±6.7)·10³ for theexperimental group receiving compound 4 in a dose of 45.5 μg/mouse,p>0.05, (87.6±4.3)·10³ for the group receiving compound 4 in a dose of91 μg/mouse, p<0.001, and (138.9±10.0)·10³ for the experimental groupreceiving compound 4 in a dose of 136.5 μg/mouse, p<0.001. Moreover,compound 4 in the dose of 136.5 μg/mouse caused significant increase oftiters of hemagglutinins in serum of immunized mice, those were 6.2±0.3for control animals and 7.5±0.2 for the group receiving compound 4 inthe dose of 136.5 μg/mouse, p<0.01 (Table 6).

The immunogenic activity of compound 4 is dose-dependent. Compound 4 inthe dose of 45.5 μg/mouse exhibits no immunogenic activity, but at thesame time in doses of 91 and 136.5 μg/mouse the compound 4 displayedhigh immunostimulating action. Thus, the relative amount of AFC on the5-th day after injection of the compound 4 increased by 70% and 233%,and total AFC amount increased by 45% and 130% accordingly in comparisonwith amount of cells in the animals immunized only with sheeperythrocytes, when a titers of hemagglutinins in serum of immunized miceraised of 21%.

TABLE 1 Effect of compound 10, injected intravenously simultaneouslywith the antigen (5 × 10⁷ SRBC/mouse, intraperitoneally), on the humoralimmune response in C57B1/6 female mice (n = 6) Amount of Amount ofantibody-forming antibody-forming cells per spleen, Amount of TreatmentSpleen cells per 10⁶ spleen Effect, % × 10³, Effect, % hemagglutininsEffect, % dose, μg index, 10⁻³ p cells, M ± SEM and p M ± SEM and p Log₂T and p None 5.9 ± 0.5 — 273.3 ± 20.4 — 31.3 ± 2.5 — 7.0 ± 0.4 — 45.56.9 ± 0.2 p > 0.05 304.9 ± 46.6 +11.6 p > 0.05 30.2 ± 5.7 −3.5 p > 0.057.8 ± 0.3 +11.4 p > 005 91.0 6.6 ± 0.4 p > 0.05 326.2 ± 65.4 +19.4 p >0.05 34.7 ± 8.5 +10.9 p > 0.05 7.8 ± 0.3 +11.4 p > 005 136.5 6.7 ± 0.2p > 0.05 651.6 ± 48.2 +138.4 p < 0.001 81.1 ± 5.8 +159.1 p < 0.001 8.5 ±0.4 +21.4 p < 0.05

TABLE 2 Effect of compound 11, injected intravenously simultaneouslywith the antigen (5 × 10⁷ SRBC/mouse, intraperitoneally), on the humoralimmune response in C57B1/6 female mice (n = 6) Amount of Amount ofantibody-forming antibody-forming cells per spleen, Amount of TreatmentSpleen cells per 10⁶ spleen Effect, % × 10³, Effect, % hemagglutininsEffect, % dose, μg index, 10⁻³ p cells, M ± SEM and p M ± SEM and p Log₂T and p None 6.5 ± 0.6 — 799.3 ± 63.3 — 101.1 ± 8.6 — 8.3 ± 0.3 — 45.56.2 ± 0.2 p > 0.05 676.8 ± 77.1 −15.3 p > 0.05  85.9 ± 8.3 −15.0 p >0.05 8.3 ± 0.3 0 91.0 6.9 ± 0.5 p > 0.05  896.8 ± 111.8 +12.2 p > 0.05 111.2 ± 11.8 +10.0 p > 0.05 8.0 ± 0.5 −3.6 p > 0.05 136.5 7.5 ± 0.4 p >0.05 1336.5 ± 59.0  +67.2 p < 0.001 182.5 ± 7.8 +80.5 p < 0.001 9.3 ±0.2 +12.0 p < 0.02

TABLE 3 Effect of compound 12, injected intravenously simultaneouslywith the antigen (5 × 10⁷ SRBC/mouse, intraperitoneally), on the humoralimmune response in C57B1/6 female mice (n = 6) Amount of Amount ofantibody-forming antibody-forming cells per spleen, Amount of TreatmentSpleen cells per 10⁶ spleen Effect, % × 10³, Effect, % hemagglutininsEffect, % dose, μg index, 10⁻³ p cells, M ± SEM and p M ± SEM and p Log₂T and p None 6.2 ± 0.5 — 291.1 ± 14.3 — 35.0 ± 3.3 — 7.2 ± 0.3 — 45.56.3 ± 0.3 p > 0.05 263.8 ± 23.6 −9.4 p >0.05 31.8 ± 2.8 −9.1 p >0.05 7.4± 0.3 +2.8 p >0.05 91.0 5.9 ± 0.3 p > 0.05 307.8 ± 30.9 +5.7 p > 0.0540.2 ± 2.7 +14.9 p > 0.05 7.4 ± 0.3 +2.8 p > 0.05 136.5 6.3 ± 0.4 p >0.05 682.6 ± 33.3 +134.5 p < 0.001 69.6 ± 6.0 +98.9 p < 0.001 7.8 ± 0.3+7.7 p > 0.05

TABLE 4 Effect of compound 8, injected intravenously simultaneously withthe antigen (5 × 10⁷ SRBC/mouse, intraperitoneally), on the humoralimmune response in C57B1/6 female mice (n = 6) Amount of Amount ofantibody-forming antibody-forming cells per spleen, Amount of TreatmentSpleen cells per 10⁶ spleen Effect, % × 10³, Effect, % hemagglutininsEffect, % dose, μg index, 10⁻³ p cells, M ± SEM and p M ± SEM and p Log₂T and p None 5.2 ± 0.2 — 501.7 ± 21.0 — 46.5 ± 2.4 — 8.5 ± 0.2 — 45.55.2 ± 0.1 — 449.1 ± 37.4 −10.5 p > 0.05 44.4 ± 4.3 −4.5 p > 0.05 8.3 ±0.3 −2.4 p > 0.05 91.0 5.1 ± 0.3 p > 0.05 592.2 ± 41.6 +18.0 p > 0.0550.9 ± 4.5 +9.5 p > 0.05 8.8 ± 0.3 +3.5 p > 0.05 136.5 5.3 ± 0.3 p >0.0S 657.8 ± 24.0 +31.1 p < 0.001 88.8 ± 8.0 +91.0 p < 0.001 8.5 ± 0.2 0

TABLE 5 Effect of compound 3, injected intravenously simultaneously withthe antigen (5 × 10⁷ SRBC/mouse, intraperitoneally), on the humoralimmune response in C57B1/6 female mice (n = 6) Amount of Amount ofantibody-forming antibody-forming cells per spleen, Amount of TreatmentSpleen cells per 10⁶ spleen Effect, % × 10³, Effect, % hemagglutininsEffect, % dose, μg index, 10⁻³ p cells, M ± SEM and p M ± SEM and p Log₂T and p None 6.2 ± 0.3 — 407.4 ± 10.7 — 40.5 ± 2.9 — 6.3 ± 0.3 — 45.56.1 ± 0.3 p > 0.05 472.2 ± 52.4 +15.9 p > 00.05 44.6 ± 3.9 +10.1 p >0.05 6.6 ± 0.2 +4.8 p > 0.05 91.0 6.2 ± 0.2 p > 0.05 827.7 ± 34.6 +103.2p < 0.001 60.3 ± 4.1 +48.9 p < 0.01 8.0 ± 0.3 +27.0 p < 0.01 136.5 6.8 ±0.3 p > 0.05 961.3 ± 56.5 +136.0 p < 0.001 107.0 ± 8.5  +164.2 p < 0.0018.7 ± 0.3 +38.1 p < 0.001

TABLE 6 Effect of compound 4, injected intravenously simultaneously withthe antigen (5 × 10⁷ SRBC/mouse, intraperitoneally), on the humoralimmune response in C57B1/6 female mice (n = 6) Amount of Amount ofantibody-forming Amount of antibody-forming cells per spleen, hema-Treatment Spleen cells per 10⁶ spleen Effect, % × 10³, Effect, %gglutinins, Effect, % dose, μg index, 10⁻³ p cells, M ± SEM and p M ±SEM and p Log₂ T and p None 6.6 ± 0.3 — 406.4 ± 16.8 — 60.4 ± 3.9 — 6.2± 0.3 — 45.5 6.6 ± 0.5 — 405.0 ± 47.9 −0.3 p >0.05 55.0 ± 6.7 −8.9 p >0.05 6.0 ± 0.3 −3.2 p > 0.05 91.0 6.3 ± 0.4 p > 0.05 692.9 ± 44.8 +70.5p < 0.001 87.6 ± 4.3 +45.0 p < 0.001 6.5 ± 0.4 +4.8 p > 0.05 136.5 6.0 ±0.5 p > 0.05 1354.3 ± 99.9  +233.2 p < 0.001 138.9 ± 10.0  +130.0 p <0.001 7.5 ± 0.2 +21.0 p < 0.01

Example 31

The Influence of Compound 13 on Humoral Immune Response

0.15 ml of compound 13 was administered intravenously to 6 female miceof the C57B1/6 line (weight 18-22 g) in doses of 45.5, 91 and 136.5 g ofcompound 13 per capita for each testing group according to the dose.Simultaneously, a suspension of 5·10⁷ sheep erytrocytes was injectedintraperitoneally (0.2 ml per capita). Mice of control group wereinjected intravenously with equal volume of saline. Compound 13 effectson humoral immune response were analyzed both by counting the quantityof AFC in the spleen according to Cunningham (per 10⁶ spleen cells andper spleen) and estimating the titers of hemagglutining antibodies inserum of the animals.

The relative amount of AFC on the 5-th day after injection was628.6±34.9 for the control animals, 608.2±61.3 for the experimentalgroup receiving compound 13 in a dose of 45.5 μg/mouse, p>0.05,525.0±97.2 for the group receiving compound 13 in a dose of 91 μg/mouse,p>0.05, and 885.9±65.8 for the experimental group receiving compound 13in a dose of 136.5 μg/mouse, p<0.01. The total AFC amount was(45.9±5.2)·10³ for control group, (48.2±6.5)·10³ for the experimentalgroup receiving compound 13 in a dose of 45.5 μg/mouse, p>0.05,(49.4±6.2)·10³ for the group receiving compound 13 in a dose of 91μg/mouse, p>0.05, and (68.2±6.9)·10³ for the experimental groupreceiving compound 13 in the dose of 136.5 μg/mouse, p<0.05 (Table 7).

The immunogenic activity of compound 13 is dose-dependent. Compound 13in doses of 45.5 and 91 μg/mouse exhibit no immunogenic activity, but atthe same time in the dose of 136.5 μg/mouse the compound 13 displayedmarked immunostimulating action. Thus, the relative amount of AFC on the5-th day after injection of the compound 13 increased by 41%, and totalAFC amount increased by 49% in comparison with amount of cells in theanimals immunized only with sheep erythrocytes.

Example 32

Compound 1a Influence on Humoral Immune Response

0.15 ml of compound 1a was administered intravenously to 6 female miceof C57B1/6 line (weight 18-22 g) in a doses of 45.5, 91 and 136.5 μg ofcompound 1a per capita for each testing group according to the dose.Simultaneously, a suspension of 5·10⁷ sheep erythrocytes was injectedintraperitoneally (0.2 ml per capita). Mice of control group wereinjected intravenously with equal volume of saline. Compound 1a effectson humoral immune response were analyzed both by counting the quantityof AFC in the spleen according to Cunningham (per 10⁶ spleen cells andper spleen) and estimating the titers of hemagglutining antibodies inserum of the animals.

The relative amount of AFC on the 5-th day after injection was772.3±47.8 for control animals, 815.8±70.9 for the experimental groupreceiving compound 1a in the dose of 45.5 μg/mouse, p>0.05, 997.2±43.4for the group receiving compound 1a in the dose of 91 μg/mouse, p<0.01,and 1176.9±58.5 for the experimental group receiving compound 1a in thedose of 136.5 μg/mouse, p<0.001. The total AFC amount was (89.9±5.9)·10³for control group, (104.6±10.7)·10³ for the experimental group receivingcompound 1a in the dose of 45.5 μg/mouse, p>0.05, (114.5±8.2)·10³ forthe group receiving compound 1a in the dose of 91 μg/mouse, p<0.05, and(149.4±9.4)·10³ for the experimental group receiving compound 1a in thedose of 136.5 μg/mouse, p<0.001 (Table 8).

Immunogenic activity of compound 1a is dose-dependent. Compound 1a inthe dose of 45.5 μg/mouse exhibits no immunogenic activity, but at thesame time in doses of 91 and 136.5 μg/mouse the compound 1a displayedmarked immunostimulating action. Thus, the relative amount of AFC on the5-th day after injection of the compound 1a increased by 29% and 52%,and total AFC amount increased by 27% and 66% correspondingly incomparison with amount of cells in the animals immunized only with sheepeyrocytes.

Example 33

Compound 14 Influence on Humoral Immune Response

0.15 ml of compound 14 was administered intravenously to 6 mice femalesof C57B1/6 line (weight 18-22 g) in a doses of 45.5, 91 and 136.5 μg ofcompound 13 per capita for each testing group according to the dose.Simultaneously suspension of 5·10⁷ sheep erythrocytes was injectedintraperitoneally (0.2 ml per capita). Mice of control group wereinjected intravenously with equal volume of saline. Compound 14 effectson humoral immune response were analyzed both by counting the quantityof AFC in the spleen according to Cunningham (per 10⁶ spleen cells andper spleen) and estimating the titers of hemagglutining antibodies inserum of the animals.

The relative amount of AFC on the 5-th day after injection was622.3±19.1 for the control animals, 554.6±52.0 for the experimentalgroup receiving compound 14 in a dose of 45.5 μg/mouse, p>0.05,645.8±64.4 for the group receiving compound 14 in a dose of 91 μg/mouse,p>0.05, and 946.1±41.9 for the experimental group receiving compound 14in a dose of 136.5 μg/mouse, p<0.001. The total AFC amount was(71.9±12.0)·10³ for the control group, (70.8±10.8)·10³ for theexperimental group receiving compound 14 in a dose of 45.5 μg/mouse,p>0.05, (80.8±12.7)·10³ for the group receiving compound 14 in a dose of91 μg/mouse, p>0.05, and (108.6±10.8)·10³ for the experimental groupreceiving compound 14 in the dose of 136.5 μg/mouse, p<0.05 (Table 9).

The immunogenic activity of compound 14 is dose-dependent. Compound 14in doses of 45.5 and 91 μg/mouse exhibits no immunogenic activity, butat the same time in the dose of 136.5 μg/mouse the compound 14 displayedmarked immunostimulating action. Thus, the relative amount of AFC on the5-th day after injection of the compound 14 increased by 52%, and totalAFC amount increased by 51% in comparison with amount of cells in theanimals immunized only with sheep erythrocytes.

Example 34

Compound 2a Influence on Humoral Immune Response

0.15 ml of compound 2a was administered intravenously to 6 mice femalesof C57B1/6 line (weight 18-22 g) in a doses of 45.5, 91 and 136.5 μg ofcompound 2a per capita for each testing group according to the dose.Simultaneously, a suspension of 5·10⁷ sheep erythrocytes was injectedintraperitoneally (0.2 ml per capita). Mice in the control group wereinjected intravenously with an equal volume of saline. The effects ofcompound 2a on humoral immune response were analyzed both by countingthe quantity of AFC in the spleen according to Cunningham (per 10⁶spleen cells and per spleen) and estimating the titers of hemagglutiningantibodies in serum of the animals.

The relative amount of AFC on the 5-th day after injection was303.3±14.4 for the control animals, 264.5±19.9 for the experimentalgroup receiving compound 2a in a dose of 45.5 μg/mouse, p>0.05,585.6±51.6 for the group receiving compound 2a in a dose of 91 μg/mouse,p<0.001, and 639.8±54.6 for the experimental group receiving compound 2ain dose of 136.5 μg/mouse, p<0.001. The total AFC amount was(40.0±3.7)·10³ for the control group, (34.0±2.6)·10³ for theexperimental group receiving compound 2a in a dose of 45.5 μg/mouse,p>0.05, (59.4±5.2)·10³ for the group receiving compound 2a in a dose of91·μg/mouse, p<0.02, and (68.3±6.1)·10³ for the experimental groupreceiving compound 2a in a dose of 136.5 μg/mouse, p<0.01 (Table 10).

The immunogenic activity of compound 2a is dose-dependent. Compound 2ain the dose of 45.5 μg/mouse exhibit no immunogenic activity, but at thesame time in doses of 91 and 136.5 μg/mouse the compound 2a displayedmarked immunostimulating action. Thus, the relative amount of AFC on the5-th day after injection of the compound 2a increased by 93% and 111%,and total AFC amount increased by 48% and 71% correspondingly incomparison with amount of cells in the animals immunized only with sheeperythrocytes.

TABLE 7 Effect of compound 13, injected intravenously simultaneouslywith the antigen (5 × 10⁷ SRBC/mouse, intraperitoneally), on the humoralimmune response in C57B1/6 female mice (n = 6) Amount of Amount ofantibody-forming Amount of antibody-forming cells per spleen, hema-Treatment Spleen cells per 10⁶ spleen Effect, % × 10³, Effect, %gglutinins, Effect, % dose, μg index, 10⁻³ p cells, M ± SEM and p M ±SEM and p Log₂ T and p None 5.6 ± 0.6 — 628.6 ± 34.9 — 45.9 ± 5.2 — 9.0± 0.5 — 45.5 5.4 ± 0.6 p > 0.05 608.2 ± 61.3 −3.2 p > 0.05 48.2 ± 6.5+5.0 p > 0.05 9.7 ± 1.2 +7,8 p > 0.05 91.0 5.7 ± 0.8 p > 0.05 525.0 ±97.2 −16.5 p > 0.05 49.4 ± 6.2 +7.6 p > 0.65 9.5 ± 1.2 +5,6 p > 0.05136.5 5.3 ± 0.4 p > 0.05 885.9 ± 65.8 +40.9 p < 0.01 68.2 ± 6.9 +48.6 p< 0.05 10.3 ± 0.9  +14,4 p > 0.05

TABLE 8 Effect of compound 1a, injected intravenously simultaneouslywith the antigen (5 × 10⁷ SRBC/mouse, intraperitoneally), on the humoralimmune response in C57B1/6 mice female (n = 6) Amount of Amount ofantibody-forming Amount of antibody-forming cells per spleen, hema-Treatment Spleen cells per 10⁶ spleen Effect, % × 10³, Effect, %gglutinins, Effect, % dose, μg index, 10⁻³ p cells, M ± SEM and p M ±SEM and p Log₂ T and p None 6.2 ± 0.4 — 772.3 ± 47.8 — 89.9 ± 5.9 — 7.7± 0.2 — 45.5 6.1 ± 0.2 p > 0.05 815.8 ± 70.9 +5.6 p > 0.05 104.6 ± 10.7+16.4 p > 0.05 7.6 ± 0.3 −1,3 p > 0.05 91.0 6.2 ± 0.2 — 997.2 ± 43.4+29.1 p < 0.01 114.5 ± 8.2  +27.4 p < 0.05 7.6 ± 0.2 −1,3 p > 0.05 136.56.9 ± 0.3 p > 0.05 1176.9 ± 58.5  +52.4 p < 0.001 149.4 ± 9.4  +66.2 p <0.001 8.2 ± 0.4 +6,5 p > 0.05

TABLE 9 Effect of compound 14, injected intravenously simultaneouslywith the antigen (5 × 10⁷ SRBC/mouse, intraperitoneally), on the humoralimmune response in C57B1/6 female mice (n = 6) Amount of Amount ofantibody-forming Amount of antibody-forming cells per spleen, hema-Treatment Spleen cells per 10⁶ spleen Effect, % × 10³, Effect, %gglutinins, Effect, % dose, μg index, 10⁻³ p cells, M ± SEM and p M ±SEM and p Log₂ T and p None 6.6 ± 0.3 — 622.3 ± 19.1 — 71.9 ± 12.0 — 7.2± 0.2 — 45.5 6.5 ± 0.2 p > 0.05 554.6 ± 52.0 −10.9 p > 0.05 70.8 ± 10.8−1.5 p > 0.05 6.5 ± 0.3 −9,7 p > 0.05 91.0 6.2 ± 0.2 p > 0.05 645.8 ±64.4 +3.8 p > 0.05 80.8 ± 12.7 +12.4 p > 0.05 7.0 ± 0.3 −2,8 p > 0.05136.5 6.5 ± 0.3 p > 0.05 946.1 ± 41.9 +52.0 p < 0.001 108.6 ± 10.8 +51.0 p < 0.05 7.2 ± 0.2 0

TABLE 10 Effect of compound 2a, injected intravenously simultaneouslywith the antigen (5 × 10⁷ SRBC/mouse, intraperitoneally), on the humoralimmune response in C57B1/6 female mice (n = 6) Amount of Amount ofantibody-forming Amount of antibody-forming cells per spleen, hema-Treatment Spleen cells per 10⁶ spleen Effect, % × 10³, Effect, %gglutinins, Effect, % dose, μg index, 10⁻³ p cells, M ± SEM and p M ±SEM and p Log₂ T and p None 7.3 ± 0.4 — 303.3 ± 14.4 — 40.0 ± 3.7 — 7.8± 0.4 — 45.5 6.4 ± 0.2 p > 0.05 264.5 ± 19.9 −12.8 p > 0.05 34.0 ± 2.6−15.0 p > 0.05 7.2 ± 0.3 −7.7 p > 0.05 91.0 6.5 ± 0.2 p > 0.05 585.6 ±51.6 +93.1 p < 0.001 59.4 ± 5.2 +48.5 p < 0.02 7.5 ± 0.2 −3.8 p > 0.05136.5 7.4 ± 0.4 p > 0.05 639.8 ± 54.6 +110.9 p < 0.001 68.3 ± 6.1 +70.8p < 0.01 8.2 ± 0.4 +5.1 p > 0.05

Example 35

The Influence of Compound 17 on Humoral Immune Response

0.15 ml of compound 17 was administered intravenously to 6 female miceof C57B1/6 line (weight 18-22 g) in doses of 45.5, 91 and 136.5 μg ofcompound 17 per capita for each testing group according to the dose.Simultaneously, a suspension of 5·10⁷ sheep erytrocytes was injectedintraperitoneally (0.2 ml per capita). A mice of control group wereinjected intravenously with an equal volume of saline. The effects ofcompound 17 on humoral immune response were analyzed both by countingthe quantity of AFC in the spleen according to Cunningham (per 10⁶spleen cells and per spleen) and estimating the titers of hemagglutiningantibodies in serum of the animals.

The relative amount of AFC on the 5-th day after injection was471.5±44.6 for control animals, 457.3±40.7 for experimental group withcompound 17 in the dose of 45.5 μg/mouse, p>0.05, 523.8±45.3 for groupwith compound 17 in the dose of 91 μg/mouse, p>0.05, and 619.6±38.9 forexperimental group with compound 17 in the dose of 136.5 μg/mouse,p<0.05. The total AFC amount was (55.2±4.9)·10³ for control group,(51.1±4.4)·10³ for experimental group with compound 17 in the dose of45.5 μg/mouse, p>0.05, (63.5±5.1)·10³ for group with compound 17 in thedose of 91 μg/mouse, p>0.05, and (71.6±4.7)·10³ for experimental groupwith compound 17 in the dose of 136.5 μg/mouse, p<0.05 (Table 11).

The immunogenic activity of compound 17 is dose-dependent. Compound 17in the doses of 45.5 and 91 μg/mouse exhibit no immunogenic activity,but at the same time in the dose of 136.5 μg/mouse the compound 17displayed marked immunostimulating action. Thus, the relative amount ofAFC on the 5-th day after injection of the compound 17 increased of 31%,and total AFC amount increased of 30% in comparison with amount of cellsin the animals immunized only with sheep erythrocytes.

Example 36

The Influence of Compound 19 on Humoral Immune Response

0.15 ml of compound 19 was administered intravenously to 6 mice femalesof C57B1/6 line (weight 18-22 g) in doses of 45.5, 91 and 136.5 μg ofcompound 19 per capita for each testing group according to the dose.Simultaneously, a suspension of 5·10⁷ sheep erythrocytes was injectedintraperitoneally (0.2 ml per capita). A mice of control group wereinjected intravenously with equal volume of saline. Compound 19 effectson humoral immune response were analyzed both by counting the quantityof AFC in the spleen according to Cunningham (per 10⁶ spleen cells andper spleen) and estimating the titers of hemagglutining antibodies inserum of the animals.

The relative amount of AFC on the 5-th day after injection was435.8±60.1 for control animals, 454.7±57.9 for experimental group withcompound 19 in the dose of 45.5 μg/mouse, p>0.05, 473.2±44.5 for groupwith compound 19 in the dose of 91 μg/mouse, p>0.05, and 612.5±47.3 forexperimental group with compound 19 in the dose of 136.5 μg/mouse,p<0.05. The total AFC amount was (46.3±5.7)·10³ for control group,(48.8±6.4)·10³ for experimental group with compound 19 in the dose of45.5 μg/mouse, p>0.05, (51.2±6.1)·10³ for group with compound 19 in thedose of 91 μg/mouse, p>0.05, and (64.7±4.9)·10³ for experimental groupwith compound 19 in the dose of 136.5 μg/mouse, p<0.05 (Table 12).

Immnunogenic activity of compound 19 is dose-dependent. Compound 19 inthe doses of 45.5 and 91 μg/mouse exhibit no immunogenic activity, butat the same time in the dose of 136.5 μg/mouse the compound 19 displayedmarked immunostimulating action. Thus, the relative amount of AFC on the5-th day after injection of the compound 19 increased of 41%, and totalAFC amount increased of 40% in comparison with amount of cells in theanimals immunized only with sheep erythrocytes.

Example 37

The Influence of Compound 20 on Humoral Immune Response

0.15 ml of compound 20 was administered intravenously to 6 female miceof C57B1/6 line (weight 18-22 g) in doses of 45.5, 91 and 136.5 μg ofcompound 20 per capita for each testing group according to the dose.Simultaneously, a suspension of 5·10⁷ sheep erythrocytes was injectedintraperitoneally (0.2 ml per capita). A mice of control group wereinjected intravenously with equal volume of saline. Compound 20 effectson humoral immune response were analyzed both by counting the quantityof AFC in the spleen according to Cunningham (per 10⁶ spleen cells andper spleen) and estimating the titers of hemagglutining antibodies inserum of the animals.

The relative amount of AFC on the 5-th day after injection was568.3±34.6 for control animals, 512.8±46.2 for experimental group withcompound 20 in the dose of 45.5 μg/mouse, p>0.05, 634.5±51.7 for groupwith compound 20 in the dose of 91 μg/mouse, p>0.05, and 845.1±43.1 forexperimental group with compound 20 in the dose of 136.5 μg/mouse,p<0.001. The total AFC amount was (50.1±3.2)·10³ for control group,(46.4±4.8)·10³ for experimental group with compound 20 in the dose of45.5 μg/mouse, p>0.05, (55.7±5.6)·10³ for group with compound 20 in thedose of 91 μg/mouse, p>0.05, and (75.8±7.7)·10³ for experimental groupwith compound 20 in the dose of 136.5 μg/mouse, p<0.02 (Table 13).

Immunogenic activity of compound 20 is dose-dependent. Compound 20 inthe doses of 45.5 and 91 μg/mouse exhibit no immunogenic activity, butat the same time in the dose of 136.5 μg/mouse the compound 20 displayedmarked immunostimulating action. Thus, the relative amount of AFC on the5-th day after injection of the compound 20 increased of 49%, and totalAFC amount increased of 51% in comparison with amount of cells in theanimals immunized only with sheep erytrocytes.

TABLE 11 The effect of compound 17, injected intravenouslysimultaneously with the antigen (5 × 10⁷ SRBC/mouse, intraperitoneally),on the humoral immune response in C57B1/6 female mice (n = 6) Amount ofAmount of antibody-forming Amount of antibody-forming cells per spleen,hema- Treatment Spleen cells per 10⁶ spleen Effect, % × 10³, Effect, %gglutinins, Effect, % dose, μg index, 10⁻³ p cells, M ± SEM and p M ±SEM and p Log₂ T and p None 5.8 ± — 471.5 ± 44.6 — 55.2 ± 4.9 — 7.6 ±0.3 — 0.3 45.5 5.8 ± — 457.3 ± 40.7 −3.0 p > 0.05 51.1 ± 4.4 −7.4 p >0.05 7.6 ± 0.2 — 0.2 91.0 5.7 ± p > 0.05 523.8 ± 45.3 +11.1 p > 0.0563.5 ± 5.1 +15.0 p > 0.05 7.8 ± 0.3 +2.6 p > 0.05 0.4 136.5 5.9 ± p >0.05 619.6 ± 38.9 +31.4 p < 0.05 71.6 ± 4.7 +29.7 p < 0.05 7.8 ± 0.4+2.6 p > 0.05 0.3

TABLE 12 The effect of compound 19, injected intravenouslysimultaneously with the antigen (5 × 10⁷ SRBC/mouse, intraperitoneally),on the humoral immune response in C57B1/6 female mice (n = 6) Amount ofAmount of antibody-forming Amount of antibody-forming cells per spleen,hema- Treatment Spleen cells per 10⁶ spleen Effect, % × 10³, Effect, %gglutinins, Effect, % dose, μg index, 10⁻³ p cells, M ± SEM and p M ±SEM and p Log₂ T and p None 6.2 ± — 435.8 ± 60.1 — 46.3 ± 5.7 — 8.0 ±0.3 — 0.2 45.5 6.2 ± — 454.7 ± 57.9 +4.3 p > 0.05 48.8 ± 6.4 +5.4 p >0.05 8.1 ± 0.3 +1,3 p > 005 0.1 91.0 6.4 ± p > 0.05 473.2 ± 44.5 +8.6p > 0.05 51.2 ± 6.1 +10.6 p > 0.05 8.3 ± 0.4 +3.8 p > 0.05 0.2 136.5 6.5± p > 0.05 612.5 ± 47.3 +40.5 p < 0.05 64.7 ± 4.9 +39.7 p < 0.05 8.5 ±0.3 +6.3 p > 0.05 0.3

TABLE 13 The effect of compound 20, injected intravenouslysimultaneously with the antigen (5 × 10⁷ SRBC/mouse, intraperitoneally),on the humoral immune response in C57B1/6 female mice (n = 6) Amount ofAmount of antibody-forming Amount of antibody-forming cells per spleen,hema- Treatment Spleen cells per 10⁶ spleen Effect, % × 10³, Effect, %gglutinins, Effect, % dose, μg index, 10⁻³ p cells, M ± SEM and p M ±SEM and p Log₂ T and p None 6.2 ± 0.3 — 568.3 ± 34.6 — 50.1 ± 3.2 — 7.4± 0.3 — 45.5 6.3 ± 0.3 p > 0.05 512.8 ± 46.2 −9.8 p > 0.05 46.4 ± 4.8−7.4 p > 0.05 7.4 ± 0.2 — 91.0 6.5 ± 0.2 p > 0.05 634.5 ± 51.7 +11.6 p >0.05 55.7 ± 5.6 +11.2 p > 0.05 7.5 ± 0.3 +1.4 p > 0.05 136.5 6.4 ± 0.4p > 0.05 845.1 ± 43.1 +48.7 p < 0.001 75.8 ± 7.7 +51.3 p < 0.02 7.6 ±0.4 +2.7 p > 0.05

Example 38

The Influence of Human AFP/Compound 3 Complex (1:100) on Humoral ImmuneResponse

0.15 ml of AFP/Compound 3 complex was administered intravenously to 6mice males of C57B1/6 line (weight 18-22 g) in the dose of 9 g of AFPand 45.5 μg of compound 3 per capita. Simultaneously suspension of 5·10⁷sheep erythrocytes was injected intraperitoneally (0.2 ml per capita).In the group with compound 3 mice were injected intravenously by one inthe dose of 45.5 μg. Animals of group with AFP received 9 μg of AFP.Mice of control group were injected intravenously with equal volume ofsaline. Complex AFP/Compound 3 effect on humoral immune response wasanalyzed by counting the quantity of AFC in the spleen according toCunningham (per 10⁶ spleen cells and per spleen).

The relative amount of AFC on the 5-th day after injection was407.4±10.7 for control animals, 362.7±31.1 for experimental group withAFP, p>0.05, 472.2±52.4 for group with compound 3, p>0.05, and716.4±49.2 for experimental group with AFP/Compound 3 complex, p<0.02.The total AFC amount was (40.5±2.9)·10³ for control group,(40.3±5.5)·10³ for experimental group with AFP, p>0.05, (44.6±3.9)·10³for group with compound 3 alone, p>0.05, and (59.9±3.7)·10³ forexperimental group with complex, p<0.05.

AFP and compound 3 alone exhibit no immunogenic activity. At the sametime AFP/Compound 3 complex (1:100) displayed marked immunostimulatingaction. Thus, the relative amount of APC on the 5-th day after injectionof the complex increased by 76%, and total AFC amount increased by 48%in comparison with amount of cells in the animals immunized only withsheep erythrocytes.

Example 39

The Influence of a Complex of Human AFP/Compound 2a (1:100) on HumoralImmune Response

0.15 ml of AFP/Compound 2a complex (1:100) was administeredintravenously to 6 mice males of C57B1/6 line (weight 18-22 g) in thedose of 9 μg of AFP and 45.5 μg of compound 2a per capita.Simultaneously, a suspension of 5·10⁷ sheep erythrocytes was injectedintraperitoneally (0.2 ml per capita). In the group with compound 2amice were injected intravenously by one in the dose of 45.5 μg. Animalsof group with AFP received 9 μg of AFP. Mice in the control group wereinjected intravenously with an equal volume of saline. The effect of theAFP/Compound 2a complex on humoral immune response was analyzed bycounting the quantity of AFC in the spleen according to Cunningham (per10⁶ spleen cells and per spleen).

The relative amount of AFC on the 5-th day after injection was303.3±14.4 for the control animals, 334.5±32.8 for the experimentalgroup receiving AFP, p>0.05, 264.5±19.9 for the group receiving compound2a alone, p>0.05, and 537.8±32.4 for the experimental group receivingAFP/Compound 2a complex, p<0.05. The total AFC amount was (40.0±3.7)·10³for the control group, (43.8±6.2)·10³ for the experimental groupreceiving AFP, p>0.05, (34.0±2.6)·10³ for the group receiving compound2a alone, p>0.05, and (57.1±4.0)·10³ for the experimental groupreceiving the complex, p<0.05.

AFP and compound 2a exhibit no immunogenic activity. At the same time acomplex of AFP/Compound 2a (1:100) displayed marked immunostimulatingaction. Thus, the relative amount of AFC on the 5-th day after injectionof the complex increased by 77%, and total AFC amount increased by 43%in comparison with amount of cells in the animals immunized only withsheep erythrocytes.

Example 40

The Influence of a Complex of HumanAFP/Compound 10 (1:200) on HumoralImmune Response

0.15 ml of AFP/Compound 10 complex (1:200) was administeredintravenously to 6 male mice of C57B1/6 line (weight 18-22 g) in thedose of 9 μg of AFP and 91 μg of compound 10 per capita. Simultaneously,a suspension of 5·10⁷ sheep erythrocytes was injected intraperitoneally(0.2 ml per capita). In the group receiving compound 10 mice wereinjected intravenously by one in the dose of 91 μg. Animals in the groupwith AFP received 9 μg of AFP. Micein the control group were injectedintravenously with an equal volume of saline. The effect of theAFP/Compound 10 complex on humoral immune response was analyzed bycounting the quantity of AFC in the spleen according to Cunningham (per10⁶ spleen cells and per spleen).

The relative amount of AFC on the 5-th day after injection was273.3±20.4 for the control animals, 275.8±12.3 for the experimentalgroup with AFP, p>0.05, 326.2±65.4 for the group with compound 10 alone,p>0.05, and 513.6±89.9 for experimental group with the complex, p<0.05.The total AFC amount was (31.3±2.5)·10³ for the control group,(33.6±2.1)·10³ for the experimental group with AFP, p>0.05,(34.7±8.5)·10³ for group with compound 10 alone, p>0.05, and(55.9±9.6)·10³ for the experimental group receiving the complex, p<0.05.

AFP and compound 10 alone exhibit no immunogenic activity. At the sametime AFP/Compound 10 complex (1:200) displayed expressedimmunostimulating action. Thus, the relative amount of AFC on the 5-thday after injection of AFP/Compound 10 complex (1:200) increased by 88%,and the total AFC amount increased by 79% in comparison with the amountof cells in the animals immunized only with sheep erythrocytes.

Example 41

The Influence of Human AFP/Compound 14 Complex (1:200) on Humoral ImmuneResponse

0.15 ml of AFP/Compound 14 complex (1:200) was administeredintravenously to 6 male mice of C57B1/6 line (weight 18-22 g) in thedose of 9 μg of AFP and 91 μg of compound 14 per capita. Simultaneously,a suspension of 5·10⁷ sheep erythrocytes was injected intraperitoneally(0.2 ml per capita). In the group with compound 14 mice were injectedintravenously by one in the dose of 91 μg. Animals of group with AFPreceived 9 μg of AFP. Mice in the control group were injectedintravenously with an equal volume of saline. The effect of theAFP/Compound 14 complex on humoral immune response was analyzed bycounting the quantity of AFC in the spleen according to Cunningham (per10⁶ spleen cells and per spleen).

The relative amount of AFC on the 5-th day after injection was622.3±19.1 for the control animals, 573.3±63.6 for the experimentalgroup receiving AFP, p>0.05, 645.8±64.4 for the group receiving compound14 alone, p>0.05, and 967.3±44.7 for the experimental group receivingthe complex, p<0.05. The total AFC amount was (71.9±12.0)·10³ for thecontrol group, (69.8±14.2)·10³ for the experimental group receiving AFP,p>0.05, (80.8±12.7)·10³ for the group receiving compound 14 alone,p>0.05, and (10.1±9.7)·10³ for the experimental group receiving thecomplex, p<0.05.

AFP and compound 14 alone exhibit no immunogenic activity. At the sametime AFP/Compound 14 complex (1:200) displayed marked immunostimulatingaction. Thus, the relative amount of AFC on the 5-th day after injectionof AFP/Compound 14 complex (1:200) increased by 55%, and total AFCamount increased by 53% in comparison with amount of cells in theanimals immunized only with sheep erythrocytes.

Example 42

The Influence of Rat AFP/Compound 7 Complex (1:200) on Humoral ImmuneResponse

0.15 ml of AFP/Compound 7 complex (1:200) was administered intravenouslyto 6 male mice of C57B1/6 line (weight 18-22 g) in the dose of 9 μg ofAFP and 91 μg of compound 7 per capita. Simultaneously, a suspension of5·10⁷ sheep erythrocytes was injected intraperitoneally (0.2 ml percapita). In the group with compound 7 mice were injected intravenouslyby one in the dose of 91 μg. Animals of group with AFP received 9 μg ofAFP. Mice of control group were injected intravenously with equal volumeof saline. AFP/Compound 7 complex effect on humoral immune response wasanalyzed by counting the quantity of AFC in the spleen according toCunningham (per 10⁶ spleen cells and per spleen).

The relative amount of AFC on the 5-th day after injection was498.6±26.3 for control animals, 521.4±49.1 for the experimental groupreceiving AFP, p>0.05, 574.9±39.6 for the group receiving compound 7alone, p>0.05, and 743.9±65.4 for the experimental group receiving thecomplex, p<0.01. The total AFC amount was (56.1±4.3)·10³ for the controlgroup, (59.7±5.1)·10³ for the experimental group receiving AFP, p>0.05,(60.4±5.4)·10³ for the group receiving compound 7 alone, p>0.05, and(82.7±9.7)·10³ for the experimental group receiving the complex, p<0.05.

AFP and compound 7 alone exhibit no immunogenic activity. At the sametime AFP/Compound 7 complex (1:200) displayed marked immunostimulatingaction. Thus, the relative amount of AFC on the 5-th day after injectionof AFP/Compound 7 complex (1:200) increased by 49%, and total AFC amountincreased by 47% in comparison with amount of cells in the animalsimmunized only with sheep erythrocytes.

Example 43

The Influence of Rat AFP/Compound 15 Complex (1:200) on Humoral ImmuneResponse

0.15 ml of AFP/Compound 15 complex (1:200) was administeredintravenously to 6 mice males of C57B1/6 line (weight 18-22 g) in thedose of 9 μg of AFP and 91 μg of compound 15 per capita. Simultaneously,a suspension of 5·10⁷ sheep erythrocytes was injected intraperitoneally(0.2 ml per capita). In the group receiving compound 15, the mice wereinjected intravenously by one in the dose of 91 μg. Animals in theAFP-group received 9 μg of AFP. Mice in the control group were injectedintravenously with an equal volume of saline. The effect of theAFP/Compound 15 complex on humoral immune response was analyzed bycounting the quantity of AFC in the spleen according to Cunningham (per10⁶ spleen cells and per spleen).

The relative amount of AFC on the 5-th day after injection was556.8±32.2 for the control animals, 507.2±27.5 for the experimentalgroup receiving AFP, p>0.05, 594.7±41.4 for the group receiving compound15 alone, p>0.05, and 837.4±69.3 for the experimental group receivingthe complex, p<0.01. The total AFC amount was (66.7±5.4)·10³ for controlgroup, (64.6±6.8)·10³ for the experimental group receiving AFP, p>0.05,(70.8±8.2)·10³ for the group receiving compound 15 alone, p>0.05, and(99.0±10.1)·10³ for the experimental group receiving the complex,p<0.05.

AFP and compound 15 alone exhibit no immunogenic activity. At the sametime AFP/Compound 15 complex (1:200) displayed marked immunostimulatingaefion. Thus, the relative amount of AFC on the 5-th day after injectionof AFP/Compound 15 complex (1:200) increased by 50%, and total AFCamount increased by 48% in comparison with amount of cells in theanimals immunized only with sheep erythrocytes.

Example 44

The Influence of Rat AFP/Compound 1 Complex (1:100) on Humoral ImmuneResponse

0.15 ml of AFP/Compound 1 complex was administered intravenously to 6mice males of C57B1/6 line (weight 18-22 g) in the dose of 9 μg of AFPand 45.5 μg of compound 1 per capita. Simultaneously, a suspension of5·10⁷ sheep erythrocytes was injected intraperitoneally (0.2 ml percapita). In the group with compound 1 mice were injected intravenouslyby one in the dose of 45.5 μg. Animals of group with AFP received 9 μgof AFP. Mice in the control group were injected intravenously with anequal volume of saline. Complex AFP/Compound 1 effect on humoral immuneresponse was analyzed by counting the quantity of AFC in the spleenaccording to Cunningham (per 10⁶ spleen cells and per spleen).

The relative amount of AFC on the 5-th day after injection was374.5±19.2 for the control animals, 346.1±26.8 for the experimentalgroup receiving AFP, p>0.05, 413.1±31.5 for the group receiving compound1, p>0.05, and 585.0±51.7 for the experimental group receivingAFP/Compound 1 complex, p<0.01. The total AFC amount was (35.8±3.6)·10³for the control group, (34.1±3.3)·10³ for the experimental groupreceiving AFP, p>0.05, (38.4±3.7)·10³ for the group receiving compound 1alone, p>0.05, and (51.2±4.1)·10³ for the experimental group receivingcomplex, p<0.05.

AFP and compound 1 alone exhibit no immunogenic activity. At the sametime AFP/Compound 1 complex (1:100) displayed marked immunostimulatingaction. Thus, the relative amount of AFC on the 5-th day after injectionof the complex increased by 56%, and total AFC amount increased by 43%in comparison with amount of cells in the animals immunized only withsheep erythrocytes.

Example 45

Preparation of a Complex of the Compounds 1 through 4, 1a through 4a,and 5 through 20, with AFP

20 mg AFP (0.3 μmol) was dissolved in 150 ml saline solution. 30 mg anycompound (1-4, 1a-4a, 5-16) was dissolved in 5 ml saline solution andwas added to the obtained AFP solution. The mixture was incubated for 30min at room temperature (20-25° C.). The obtained complex wasconcentrated to 10 ml using Sartocon® Micro Unit (Sartorius) forhigh-molecular weight compounds with a 20 000 Da membrane cut off. Thefinal solution was sterilized with a syringe tip Minisart®-SRP SyringeFilter (Sartorius) a membrane of 22μ pore size. A sterilizedconcentrated preparation was distributed in 10 vials of 1 ml each. Vialswere blown with argon stream, closed firmly and stored at 4-8° C.

Example 46

AFP Binding with Compounds 1-4, 1a-4a, and 5-20

In order to determine the affinity human of AFP to compounds 1 through4, 1a through 4a, and 5 through 20, a competitive substitution of[5,6,8,9,11,12,14,15-³H] arachidonic acid from the protein's bindingsite was used. To tubes containing 0.05 mmol AFP in 1 ml 0.1 Mbicarbonate buffer and 0.7 pmol [³H]arachidonic acid and increasingamounts (5-5000 pmol) of arachidonic acid or any one of the titlecompounds 1-4, 1a-4a, and 5-20 were added. The tubes were incubated for2 hr at room temperature. To separate protein-bound and free fractionsof [³H] arachidonic acid, tubes were incubated at 4° C. for 15 min withdextran covered activated carbon (0.5% suspension). The carbon wassedimented by centrifugation at 3000×g, aliquotes were added to 10 mlscintillating mixture and the vials were measured in a beta-counter.

The binding parameters of arachidonic acid and the compounds (1-4,1a-4a, 5-16) the number of binding sites per protein molecule werecalculated according to Scatchard (Scatchard O., Ann.N.Y.Acad.Sci. 51.,p 660-664, 1949), and Blondeau (Blondeau J.-P., et al., Steroids, V. 32,N 5, P. 563-575, 1978).

Based on three independent determinations; the K_(a) value for AFP witharachidonic acid was found to be 6·10⁷ M⁻¹ and n>1.2. For the titlecompounds 1 through 4, 1a through 4a, and 5 through 20, the inhibitionequilibrium association constants (K_(i)) were in the interval of0.9·10⁶ M⁻¹-4·10⁶ M⁻¹.

Example 47

Determination the Molecular Weight of Complexes Human AFP with TitleCompounds

In order to determine the molar ratio between AFP and the compounds 1-4,1a-4a, and 5-20 in complexes, ultrafiltration and/or gel-filtrationchromatography was performed. The detection of complexes was carried outby measuring the absorbance at 280 nm and 345 nm and counting todetermine, if the radioactive label [¹²⁵I] had been incorporated intothe AFP molecule and [³H] into compounds 5-8 ([³H] arachidonic acid wasused in synthesis).

The present inventor carried out gel exclusion chromatography of AFPcomplexes with compounds 1-4, 1a-4a, and 5-20 at different amounts ofcomponents—1 mole AFP per 700, 1400 and 2100 moles of the compound, forestimation of the AFP maximum binding capacity. The data obtainedindicate that the ratio AFP/compound in complexes is between 1/100 and1/300. In addition, these molar ratios AFP/compound in the complexesdepend on the initial amounts of compounds in the solution. Thus,initial amounts of 1 mole AFP per 700 moles of the compound gives aratio close to 1/100 (AFP/compound) in the complex; amounts of 1 moleAFP per 1400 moles of the compound gives a ratio close to 1/200(AFP/compound) in the complex; amounts of 1 mole AFP per 2100 moles ofthe compound gives a ratio close to 1/300 (AFP/compound) in the complex.

The synthesis scheme can be outlined as below:

where R is:

where R is:

where R is

Although the invention has been described with regard to its preferredembodiments, which constitute the best mode presently known to theinventor, it should be understood that various changes and modificationsas would be obvious to one having the ordinary skill in this art may bemade without departing from the scope of the invention which is setforth in the claims appended hereto.

What is claimed is:
 1. A complex of alpha-fetoprotein (AFP) and acompound having the following formula R—CONH—X—PO(OH)₂ wherein R isindependently selected from

and CH₃(CH₂)₄(CH═CH—CH₂)₄CH₂CH₂— and CH₃(CH₂CH═CH)₆CH₂CH₂— andCH₃(CH₂CH═CH)₅(CH₂)₃— and CH₃(CH₂CH═CH)₃(CH₂)₇— and where X isindependently selected from —CH₂—CH₂— and —CH(CO₂H)—CH₂— and—CH(CO₂H)—CH(CH₃— and —CH(CO₂H)—CH₂—C₆H₅—.
 2. A complex comprisingalpha-fetoprotein and a compound selected from the group consisting of:N-(all-trans-retinoyl)-o-phospho-2-aminoethanol,N-(13-cis-retinoyl)-o-phospho-2-aminoethanol,N-(all-trans-retinoyl)-o-phospho-L-serine,N-(13-cis-retinoyl)-o-phospho-L-serine,N-(all-trans-retinoyl)-o-phospho-L-threonine,N-(13-cis-retinoyl)-o-phospho-L-threonine,N-(all-trans-retinoyl)-o-phospho-L-tyrosine, andN-(13-cis-retinoyl)-o-phospho-L-tyrosine.
 3. A complex comprisingalpha-fetoprotein and a compound selected from the group consisting of:N-linolenoyl-o-phospho-2-aminoethanol, N-linolenoyl-o-phospho-L-serine,N-linolenoyl-o-phospho-L-threonine, andN-linolenoyl-o-phospho-L-tyrosine.
 4. A complex comprisingalpha-fetoprotein and a compound selected from the group consisting of:N-docosahexaenoyl-o-phospho-2-aminoethanol,N-docosahexaenoyl-o-phospho-L-serine,N-docosahexaenoyl-o-phospho-L-threonine, andN-docosahexaenoyl-o-phospho-L-tyrosine.
 5. A complex comprisingalpha-fetoprotein and a compound selected from the group consisting of:N-eicosapentaenoyl-o-phospho-2-aminoethanol,N-eicosapentaenoyl-o-phospho-L-serine,N-eicosapentaenoyl-o-phospho-L-threonine, andN-eicosapentaenoyl-o-phospho-L-tyrosine.
 6. A complex comprisingalpha-fetoprotein and a compound selected from the group consisting of:N-arachidonoyl-o-phospho-2-aminoethanol,N-arachidonoyl-o-phospho-L-serine, N-arachidonoyl-o-phospho-L-threonine,and N-arachidonoyl-o-phospho-L-tyrosine.
 7. A complex according to claim1 wherein the complex is an equilibrium reversible complex wherein theconcentration of said compound is at least equal to the critical micelleconcentration of said complex.
 8. A complex according to claim 2 whereinthe complex is an equilibrium reversible complex wherein theconcentration of said compound is at least equal to the critical micelleconcentration of said complex.
 9. A complex according to claim 3 whereinthe complex is an equilibrium reversible complex wherein theconcentration of said compound is at least equal to the critical micelleconcentration of said complex.
 10. A complex according to claim 4wherein the complex is an equilibrium reversible complex wherein theconcentration of said compound is at least equal to the critical micelleconcentration of said complex.
 11. A complex according to claim 5wherein the complex is an equilibrium reversible complex wherein theconcentration of said compound is at least equal to the critical micelleconcentration of said complex.
 12. A complex according to claim 6wherein the complex is an equilibrium reversible complex wherein theconcentration of said compound is at least equal to the critical micelleconcentration of said complex.
 13. A complex according to claim 1wherein the complex is an equilibrium reversible complex wherein theconcentration of said compound is 100 to 300 moles per molealpha-fetoprotein in said complex.
 14. A complex according to claim 2wherein the complex is an equilibrium reversible complex wherein theconcentration of said compound is 100 to 300 moles per molealpha-fetoprotein in said complex.
 15. A complex according to claim 3wherein the complex is an equilibrium reversible complex wherein theconcentration of said compound is 100 to 300 moles per molealpha-fetoprotein in said complex.
 16. A complex according to claim 4wherein the complex is an equilibrium reversible complex wherein theconcentration of said compound is 100 to 300 moles per molealpha-fetoprotein in said complex.
 17. A complex according to claim 5wherein the complex is an equilibrium reversible complex wherein theconcentration of said compound is 100 to 300 moles per molealpha-fetoprotein in said complex.
 18. A complex according to claim 6wherein the complex is an equilibrium reversible complex wherein theconcentration of said compound is 100 to 300 moles per molealpha-fetoprotein in said complex.
 19. A pharmaceutical compositioncomprising at least one complex according to claim
 1. 20. Apharmaceutical composition comprising at least one complex according toclaim
 2. 21. A pharmaceutical composition comprising at least onecomplex according to claim
 3. 22. A pharmaceutical compositioncomprising at least one complex according to claim
 4. 23. Apharmaceutical composition comprising at least one complex according toclaim
 5. 24. A pharmaceutical composition comprising at least onecomplex according to claim
 6. 25. A method for the treatment of cancercomprising administering a complex according to claim 1 to a patient.26. A method for the treatment of cancer comprising administering acomplex according to claim 2 to a patient.
 27. A method for thetreatment of cancer comprising administering a complex according toclaim 3 to a patient.
 28. A method for the treatment of cancercomprising administering a complex according to claim 4 to a patient.29. A method for the treatment of cancer comprising administering acomplex according to claim 5 to a patient.
 30. A method for thetreatment of cancer comprising administering a complex according toclaim 6 to a patient.